Author response:
The following is the authors’ response to the original reviews.
Public Reviews:
Reviewer #1 (Public review):
This study by Vitar et al. probes the molecular identity and functional specialization of pH-sensing channels in cerebrospinal fluid-contacting neurons (CSFcNs). Combining patch-clamp electrophysiology, laser-based local acidification, immunohistochemistry, and confocal imaging, the authors propose that PKD2L1 channels localized to the apical protrusion (ApPr) function as the predominant dual-mode pH sensor in these cells.
The work establishes a compelling spatial-physiological link between channel localization and chemosensory behavior. The integration of optical and electrical approaches is technically strong, and the separation of phasic and sustained response modes offers a useful conceptual advance for understanding how CSF composition is monitored.
Several aspects of data interpretation, however, require clarification or reanalysis-most notably the single-channel analyses (event counts, Po metrics, and mixed parameters), the statistical treatment, and the interpretation of purported "OFF currents." Additional issues include PKD2L1-TRPP3 nomenclature consistency, kinetic comparison with ASICs, and the physiological relevance of the extreme acidification paradigm. Addressing these points will substantially improve reproducibility and mechanistic depth.
Overall, this is a scientifically important and technically sophisticated study that advances our understanding of CSF sensing, provided that the analytical and interpretative weaknesses are satisfactorily corrected.
(1) The authors should re-analyze electrophysiological data, focusing on macroscopic currents rather than statistically unreliable Po calculations. Remove or revise the Po analysis, which currently conflates current amplitude and open probability.
We agree with the reviewer that the Po analysis has strong limitations, particularly in experiments where the recording times are short, like when extracellular pH is changed either by photolysis (Figure 4D) or puff applications (Figure 3Aa). In order to circumvent that problem and not to rely only on Po estimations, we used alternative methods as well, including the analysis of the current membrane charge that we have used extensively during the manuscript (Figures 3A and 4D, for example) or the analysis of the event latencies (Figure 4G). Nevertheless, single-channel recordings clearly contain information that is not included in the macroscopic current analysis. We intend to stress in the revised version that the elementary current amplitude is conserved by manipulations such as pH changes, leaving the total number of channels (N) and the channel open probability (Po) as possible culprits for the current changes. Since these changes are rapid and reversible, it is likely that N stays constant and that Po changes. In order to address the reviewer’s concern, we propose the following changes/reanalysis: i) to report in each condition the minimum N (maximum observed openings; for example, in Figure 3Aa the minimum N goes from 4 in control conditions to 1 during the puff of the pH 6.4 solution). This method (to estimate N by counting the maximum number of open channels), while imperfect, provides a tentative estimate of Po; ii) following the previous point, we propose to reword the text (and images) and use the expression “apparent Po” instead of “Po”; iii) to report the fraction of time that the channels remain open. Also, we acknowledge that some traces are confusing (Figure 3Aa, top) as they seem to show macroscopic currents. We will modify those figures by plotting the amplitude histograms (as in Figure 1Bb) in order to show unambiguously that current recordings from CSFcNs only show single-channel activities.
(2) PKD2L1-TRPP3 nomenclature should be clarified and all figure labels, legends, and text should use consistent terminology throughout.
We agree with the reviewer that the nomenclature concerning polycystin members is confusing. In this manuscript we have followed the nomenclature that has been proposed in a recent, comprehensive review on polycystin channels by Palomero, Larmore and DeCaen (Palomero et al. 2023), where the authors refer to the channels by their gene name. In this review, the authors indicate that PKD2L1 channels correspond to TRPP2 (formerly TRPP3, their table 1). In another, recent review on TRP channels, however, the authors refer to the PKD2L1 channel as TRPP3 (Zhang et al. 2023). In order to avoid any confusion we will remove from the text any reference to the TRPP nomenclature and stick to the PKD2L1 name.
(3) The authors should reinterpret the so-called OFF currents as pH-dependent recovery or relaxation phenomena, not as distinct current species. Remove the term "OFF response" from the manuscript.
We concur with the reviewer that the term “OFF response” is not very helpful from the biophysical perspective and conveys the idea that it is another current. We will remove the term “OFF response” or “OFF current” in the revised manuscript and replace it by the term “photolysis-evoked PKD2L1 current”. Also, we will condense two sections (“The proton-induced current is an off-current” and “The off-current is mediated by the activation of PKD2L1 channels”) into a single new section (“The photolysis-induced current is mediated by PKD2L1 channels”), as separating the description of the photolysis-evoked PKD2L1 current compromises its description. Finally, we will rewrite the discussion to better describe this current.
(4) Evidence for physiological relevance should be provided, including data from milder acidification (pH 6.5-6.8) and, where appropriate, comparisons with ASIC-mediated currents to place PKD2L1 activity in context.
This is partly addressed in Figure 3. The data there indicates that PKD2L1 channels are very sensitive to pH variations around physiological pH. In order to make this conclusion stronger, we will add to the figure the EC50 values drawn from the fittings. In terms of the ASIC-mediated currents, one of our main conclusions is that ASIC channels are not present in the ApPr, as the effects of proton photolysis in the ApPr and are not blocked by ASIC channels blockers. Our results indicate that PKD2L1 channels are the exclusive pH sensitive channels in the ApPr, while ASIC channels are probably the acid-sensitive channels in the soma, although we have not studied the latter in detail. Following this and the editor’s comments, the subsection “the involvement of ASICs” in the Discussion has been modified.
(5) Terminology and data presentation should be unified, adopting consistent use of "predominant" (instead of "exclusive") and "sustained" (instead of "tonic"), and all statistical formats and units should be standardized.
Following the suggestions of the reviewer, an exhaustive work will be performed to unify terminology, data presentation and correct the text following the reviewer’s suggestions.
(6) The Discussion should be expanded to address potential Ca2+ -dependent signaling mechanisms downstream of PKD2L1 activation and their possible roles in CSF flow regulation and central chemoreception.
This is indeed a very interesting and currently unresolved point in the physiology of CSFcNs. Published data indicate that calcium flowing into the cell through PKD2L1 channels is a key regulator of apical process physiology: on the one hand, PKD2L1 channels are calcium permeable and at the same time, they are inhibited by intracellular calcium (DeCaen et al. 2016). Also, ultrastructural data indicate that the ApPr is rich in mitochondria and tubulo-vesicular structures resembling the Golgi apparatus (Bjugn et al. 1988; Bruni et Reddy 1987), which are intracellular organelles that contribute to calcium homeostasis. Altogether, this evidence suggests that intraApPr calcium concentration needs to be finely regulated, both in space and in time, in order for the ApPr to fulfil its physiological roles. Based on what has been published in the literature, we can speculate that these calcium signals can be decoded by several systems: i) calcium can act as the second messenger linking the activation of the multimodal PKD2L1 channels to changes in CSFcNs excitability, which in turn regulate spinal neuronal networks controlling locomotor activity; ii) calcium could initiate neurosecretion of different molecules from the ApPr to the central canal (as has been proposed by the Wyart group in the zebrafish in the context of bacterial infections(Prendergast et al. 2023)); iii) calcium could activate the Hedgehog signaling pathways (as has been shown by (Delling et al. 2013)); iv) calcium could modulate CSF flow directly (by modulating ciliary activity) or indirectly (by modulating ependymal cells ciliary activity through paracrine interactions). Resolving these downstream pathways is essential to fully define the role of CSFcNs as integrators of CSF homeostasis. We will expand this issue in the Discussion of the revised ms.
Reviewer #2 (Public review):
Summary:
Cerebrospinal fluid contacting neurons (CSF-cNs) are GABAergic cells surrounding the spinal cord central canal (CC). In mammals, their soma lies sub-ependymally, with a dendritic-like apical extension (AP) terminating as a bulb inside the CC.
How this anatomy-soma and AP in distinct extracellular environments relate to their multimodal CSF-sensing function remains unclear.
The authors confirm that in GATA3:GFP mice, where these cells are labeled, that CSFcNs exhibit prominent spontaneous electrical activity mediated by PKD2L1 (TRPP2) channels, non-selective cation channels with ~200 pS conductance modulated by protons and mechanical forces.
They investigated PKD2L1 pH sensitivity and its effects on CSFcN excitability. They uncovered that PKD2L1 generates both phasic and tonic currents, bidirectionally modulated by pH with high sensitivity near physiological values.
Combining electrophysiology (intact and isolated AP recordings) with elegant laser-photolysis, they show that functional PKD2L1 channels localize specifically to the apical extension (AP).
This spatial segregation, coupled with PKD2L1's biophysical properties (high conductance, pH sensitivity) and the AP's unique features (very high input resistance), renders CSFcN excitability highly sensitive to PKD2L1 modulation. Their findings reveal how the AP's properties are optimised for its sensory role.
Strengths:
This is a very convincing demonstration using elegant and challenging approaches (uncaging, outside out patch of the AP) together to form a complete understanding of how these sensory cells can detect the changes of pH in the CSF so finely.
Weaknesses:
The following do not constitute weaknesses; rather, they are minor requests that this reviewer considers would complete this beautiful study.
(1) It would be nice to quantify further the relation in spontaneous as well as in acidic or basic pH between the effects observed on channel opening and holding current: do they always vary together and in a linear way?
Following the reviewer’s suggestion, we have performed a Spearman’s rank correlation test, which shows a significant correlation between the changes in the apparent open probability and holding current (paired experiments; ctrl vs pH 6.4 pressure applications; p < 0.05, Spearman r = 0.72 and critical value = 0.67). The Pearson correlation coefficient calculated on the same data set = 0.63 and the critical value is 0.632, which indicates that the correlation is not linear. We will add this analysis to the manuscript.
(2) Since CSF-cNs also respond to changes in osmolarity (Orts Dell Immagine 2013) & mechanosensory stimulations in a PKD2L1 dependent manner (Sternberg NC 2018), it would be nice to test the same results whether the same results hold true on the role of PKD2L1 in AP for pressure application of changes in osmolarity.
This is a very important point. As the reviewer mentions, previously published experimental evidence indicates that CSFcNs are also sensitive to osmolarity changes and mechanical stimulation in a PKD2L1-dependent manner. It is therefore reasonable to assume that, as for the pH sensitivity, osmotic and mechanical sensitivity depends on channels segregated to the ApPr. For the mechanosensitivity, the spatial segregation could be tested by “touching” either the ApPr or the soma with a piezo-controlled blunted pipette (see, for example, Hao et al. 2013). However, the sensitivity to osmotic changes is much more difficult to assess, as pressure application does not have enough spatial resolution to discriminate among compartments in such a compact cell such as the CSFcNs. In theory, a highly spatially localized osmotic jump could be reached with photolysis, but a caged compound releasing many osmotic particles simultaneously should be used. In typical photolysis experiments, a localized osmotic jump is produced, but it is very low (in the order of 1 to 2 mOsm).
In mice, like in fish (Sternberg et al, NC 2018), we can observe throughout the figures that a large fraction of the channel activity occurs with partial and very fast openings of the PKD2L1 channel. I recommend the authors analyse the points below:
(a) To what extent do these partial openings of the channel contribute to the changes in holding current and resting potential?
As the reviewer indicates, these partial and very fast openings are a characteristic of PKD2L1 single-channel activity that seems to be present in different species. However, estimating what is the exact contribution of these events to the sustained current would require a detailed model of the channel that it is still lacking. Indeed, the exact mechanism by which CSFcNs show this prominent sustained current is unknown and should definitely being addressed in future works.
(b) In the trace from the outside out AP, it looks like the partial transient openings are gone. Can the authors verify whether these partial openings are only present in somatic recordings?
The outside-out recordings from the ApPr also show some partial openings (please look at the upper trace in Figure 4Db). We will specifically mention this important point in the revised version of the ms.
(3) Previous studies have observed expression of metabotropic Glutamate receptors in CSF-cNs (transcriptome from Prendergast et al CB 2023). The authors only used blockers for ionotropic glutamate receptors in their recordings: could it be that these metabotropic receptors influence the response to uncaging of MNI-Glu when glutamate is co-released with a proton?
We thank the reviewer for pointing out the presence of metabotropic glutamate receptors in CSFcNs. However, our evidence indicates that there is no contribution of metabotropic receptors when uncaging MNIglutamate because: i) the response obtained when uncaging MNI-gLGG (where there is no glutamate release; Figure 5Ab) and ii) the response obtained when uncaging protons from DPNIGABA (a GABA cage that has similar photochemistry than MNI cages which also release a proton when photolysed; data not shown), are the same. Indeed, in both experiments (MNI-gLGG or DPNI-GABA uncaging) a clear photolysis-evoked PKD2L1 current can be observed.
(4) In the outside out patch of the AP, PKD2L1 unitary currents appear rare. Could it be that the disruption in the cilium or underlying actin/myosin cytoskeleton drastically alter the open probability of the channel?
Although we have not quantified it, the reviewer is right that the opening frequency of PKD2L1 channels in the outside-out patches is lower than in the whole-ApPr recordings. We interpreted this difference as a difference in channel number. However, another plausible interpretation is that, as the reviewer suggests, the biophysics of the channels are affected because the protein is taken out from its normal ionic environment and/or loses important interactions with regulatory proteins.
(5) Could the authors use drugs against ASIC to specify which ASIC channels contribute to the pH response in the soma?
As described in the manuscript, we did perform experiments with ASIC channel blockers, although we did not attempt to characterize the specific ASIC channel involved in the somatic response. Based on what has been published in the literature, we used both psalmotoxin-1 (which blocks ASIC1 channels) and APETx2 (which blocks ASIC3 channels). The presence of ASIC1 channels in mice CSFcNs has been shown by (Orts-Del’Immagine et al. 2012; Orts-Del’Immagine et al. 2016), while the presence of ASIC3 in the lamprey CSFcNs has been shown by (Jalalvand et al. 2016). When we puff an acidic solution aiming at the soma, we can record an inward current that is blocked by psalmotoxin-1, although there is always a small component remaining (as originally shown by Orts-Del’Immagine in the aforementioned articles); however, we have not attempted to block this small component that remains after psalmotoxin-1 bath application.
(6) This is out of the scope of this study, but we did observe in fish a very rarely-opening channel in the PKD2L1KO mutant. I wonder if the authors have similar observations in the conditions where PKD2L1 is mainly in the closed state.
We have never seen such kind of openings in our recordings (when the channel is closed or in the presence of dibucaine).
Bjugn, R, H K Haugland, et P R Flood. 1988. “Ultrastructure of the mouse spinal cord ependyma.” Journal of Anatomy 160 (octobre): 117‑25.
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DeCaen, Paul G., Xiaowen Liu, Sunday Abiria, et David E. Clapham. 2016. “Atypical Calcium Regulation of the PKD2-L1 Polycystin Ion Channel”. eLife 5 (juin): e13413. https://doi.org/10.7554/eLife.13413.
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Recommendations for the authors:
Reviewing Editor Comments:
Both reviewers were very impressed with your work and definitely feel it is a scientifically important and technically sophisticated study that advances our understanding of CSF sensing. They, however, request some re-analysis of the data and discussion with minimum new experiments, if any. I think, if feasible, this will improve the quality of the study and would look forward to receiving a revised version.
Reviewer #1 (Recommendations for the authors):
(1) Figure 1 - Molecular identity and localization of PKD2L1
Major
Nomenclature clarity.
Please clarify the distinction between PKD2L1 and TRPP3. Several parts of the text and figure labels appear to conflate these names. PKD2L1 corresponds to the TRPP3 subfamily member and should not be interchanged with PKD2/TRPP2. Please confirm and update the nomenclature consistently throughout the manuscript (text, figure labels, and captions).
We have addressed this issue in response to reviewer 1. There seems to be some confusion in the literature concerning the nomenclature of PKD2L1 channels as in some recent publications the PKD2L1 channels are still named as TRPP3. However, the nomenclature of PKD2L1 channels or TRPP2, was updated in 2016 (Wu, Sweet and Clapham, Pharmacological Reviews, 2010). As indicated in response to reviewer 1, we have removed from the text any reference to the TRPP nomenclature and stuck to the PKD2L1 name.
Physiological meaning of apical restriction.
Expand the discussion of why apical-restricted localization matters. Specifically, address how segregation to the ApPr could support directional sensing of CSF flow and/or detection of localized pH gradients.
Following the reviewers and editor’s comments, we have revised the discussion in order to take this and other comments into account.
Open-state annotation (O3).
Please include the O3 state in panels Ba and Bb; the figure clearly shows an additional open level consistent with O3.
The editor is right in that there is another state that presumably corresponds to O3. Following the editor’s recommendation, we now indicate this 3rd level and add a short sentence explaining this in figure 1 legend.
Minor
Indicate the ROI definition and background-subtraction method used for fluorescence quantification (ApPr vs soma).
Not applicable for this figure.
Ensure the same intensity scale (lookup table and range) is used across panels to enable direct comparison.
Done.
(2) Figure 2 - Electrophysiological characterization of ApPr and somatic recordings Major
Definition of "PKD2L1-dependent current."
Define this term precisely at its first appearance. Specify whether it denotes currents inhibited by dibucaine, abolished in PKD2L1-knockout preparations, or both.
Done
Statistical power of single-channel analysis.
The number of observed openings (< 1000 events) is too low to estimate open probability (Po) reliably. Please re-analyze the data using macroscopic current traces rather than Po-based kinetics.
Confounded Po analysis.
The current Po analysis mixes current amplitude and Po in the same calculation, conflating independent variables. Re-evaluate or remove this analysis.
Unknown channel count.
Because the number of channels in each patch is unknown, Po and "closed probability" values cannot be interpreted meaningfully. Focus instead on the averaged macroscopic current density.
General analytical validity.
The single-channel analyses in Figure 2 are not interpretable under these experimental conditions. Closed-time distributions and Po-based metrics (e.g., "Po1," "P2") depend critically on channel number and event sampling. Moreover, the manuscript applies essentially the same Po methodology across conditions (Po1 vs P2), which adds no mechanistic resolution and risks circular interpretation.
Actionable recommendation:
Remove Po- and closed-time-based analyses from Figure 2 and from the manuscript as a whole. Reanalyze the data using metrics that remain valid when the channel number is unknown:
Macroscopic current analysis (leak-subtracted current density, I-V relationships, activation time constants).
Single-channel conductance only (amplitude histograms and unitary slope conductance), without attempting Po or dwell-time inference.
Report filtering bandwidth and sampling rate, and restrict statistical treatment to these robust parameters.
Following the reviewers (see above) and editors’ recommendations, we have reanalyzed the data in order to avoid the analysis based on Po and Pc. We have instead calculated from the recordings other 2 parameters, nmax (the maximum number of channels that open simultaneously during a 500 ms time window) and the total open time of a single channel during the same 500 ms time window. The main text, figures and corresponding figure legends, and the Materials and Methods section have been changed accordingly. Notably, the Po and Pc analysis were removed from Figures 3, 4 and Supplementary Figure 3, and replaced by the above-mentioned parameters. Also, the fact that the recordings are not long enough to calculate Po is now specifically mentioned in the Materials and Methods section, lines 785 to 790. In addition, the analysis in Figure 3Ce has been redone so that the activity of the channel as a function of pH is now plotted as the normalized apparent Po (relative to the apparent Po value at pH 7.4).
Minor
State whether input-resistance values (1.8-4.4 GΩ) were leak-subtracted and series-resistance-compensated.
As already mentioned in the methodology section (line 693), series resistance was not compensated for during the experiments. We have now added a sentence in the methodology section indicating that in the voltage range that was chosen for the analysis of the input resistance, no voltage-dependent conductance was activated (lines 809 to 812).
Ensure unit consistency: use Po or normalized Po rather than frequency (Hz) throughout.
(3) Figure 3 - pH-evoked currents and kinetics
Major
Invalid Po analysis (Fig. 3Ca-Ce).
The Po- and Pc-based single-channel analyses in panels 3Ca-3Ce should be deleted. As noted earlier, the event count is insufficient, and the number of active channels in each patch is unknown. Under these conditions, Po and Pc values have no quantitative meaning and could mislead readers. These panels do not contribute additional mechanistic insight beyond the macroscopic current data and therefore, should be removed. If retained for illustrative purposes, they must be explicitly labeled as representative traces without any statistical quantification.
As we mentioned above, we removed the Po and Pc analysis from the manuscript.
Minor
Present regression equations and r2 values for the linear fits shown in Fig. 3D.
Done (lines 947951).
Confirm that all axes include units and identical scaling between conditions for direct comparison.
Done.
(4) Figure 4 - Laser photolysis and local stimulation experiments
Major
Laser timing annotation.
Clearly mark laser-pulse timing (e.g., arrow or shaded region) on all current traces to facilitate interpretation.
We thank the editor for pointing out the inconsistencies in terms of the laser pulse timing. To indicate the laser pulses, we have now added an arrowhead in cases where a single sweep is shown (for example, Figure 3D), and an arrowhead and a dotted magenta vertical line in cases where multiple sweeps are shown (for example, Figure 3C).
pH calibration within the laser spot.
Provide quantitative calibration of pH changes induced by laser photolysis, including information on spot size, local diffusion, and estimated pH recovery kinetics.
This is an important point and we thank the editor for mentioning it. We have now completed the subsection untitled “photolysis” where we provide information on the lateral and axial dimensions of the photolysis laser spot used in this work (lines 734 to 737). We have also rewritten part of Figure 5A legend to highlight the fact that the experiments presented there (photolysis on top of the ApPr and next to it) are compatible with a high spatial resolution of proton release (lines 1023 to 1026).
On the other hand, we have attempted to perform pH calibrations in the setup using the pH-sensitive dye pyranine (or HPTS: 8-Hydroxypyrene-1,3,6-trisulfonic acid). HPTS is a very useful tool for pH calibrations in the physiological range: its pKa value is close to 7.2, and it can be used as a ratiometric dye (its fluorescence is pH-independent at 405–410 nm and pH-dependent at 450 nm). Unfortunately, when trying to perform a calibration under the conditions of a real experiment,
where photolysis occurs in a tiny volume (approximately 1 µm³ in a total bath volume of more than 1 ml), we encountered the following problem, which made it impossible to obtain any useful data: the 405 nm uncaging pulse bleaches the dye, and any useful information is lost. Also, our imaging system is not fast enough to follow the pH change. As it is discussed in the Materials and Methods section, subsection “Estimation of the pH drop induced by photolysis” (line 814), the fast protonation of bicarbonate indicates that the pH change induced by the photolysis recovers in the submillisecond time range.
Repeated stimulation effects.
Discuss whether repeated photolysis induces adaptation or desensitization of PKD2L1 currents, and indicate whether current amplitude decreases across successive trials.
This issue is now specifically mentioned in the Materials and Methods section, lines 739 to 741.
Invalid interpretation of the "OFF response."
The interpretation of the so-called "OFF response" in Figure 4C is not supported by the presented data. There is no evidence for a bona fide OFF current, and the literature cited does not demonstrate such a phenomenon for PKD2L1 alone. Rather, previous studies implicate PKD1L3-dependent mechanisms in similar biphasic responses. Please reconsider the cited references and remove claims of an OFF current attributed to PKD2L1.
Done.
Actionable recommendations:
Do not use the term "OFF response" throughout the manuscript. Recast these transients as pH dependent recovery or relaxation of current following cessation of acidification.
Done. We have performed extensive rewriting and reorganization of the Results and Discussion in order to take into account both the reviewer’s and editor’s comments. Please also take a look at comment #3 of Reviewer 1 and point 11 below.
Include continuous-illumination controls (sustained local acidification) to test whether a steady state current is maintained. This will clarify whether the post-stimulus transient reflects recovery kinetics rather than a distinct current species.
We thank the editor for suggesting this experiment. However, continuous laser illumination is a difficult manipulation and does not necessarily lead to an acidification of the illuminated volume. Indeed, with continuous illumination the cage is lost from the illumination spot and needs to be replaced by diffusion from the non-illuminated volume, leading to non-homogeneous concentrations. Also, the chances of inducing photo damage are higher. We thus designed a similar experiment where instead of performing continuous illumination we photolysed with short and high frequency trains in order to produce a long-lasting acidification. The results of these experiments have been added to the manuscript as part of the results section and in Figure 5H. Similarly to what is seen with single illuminations, the photolysis trains induce a current that appears almost exclusively at the end of the train, implying that the current is indeed a PKD2L1-dependent recovery current.
Align the current time course with measured or estimated local pH (or calibrated proxy) to demonstrate causal coupling and avoid implying a separate conductance.
We have added the calculated pH change to the inset of Figure 4C as an example.
Revise the schematic/model figure and textual description accordingly, restricting the framework to phasic vs sustained activation modes without invoking a separate OFF current for PKD2L1.
Done.
Minor
Include scale bars, sample numbers (n), and laser parameters (duration, power) in all panels.
In order not to make the figure and the panels very heavy in the original version, we tried to limit the number of scale bars. We have now performed some modifications, added the missing scale bars, and changed the figure legend in order to take into account the editor’s comments. We have also corrected a few values that were wrongly reported.
Standardize p-value formatting (e.g., p = 6 × 10 ⁶) throughout the figure and legend.
Done.
(5) Figure 5 - Single-channel recordings
Major
Mixed parameters (current amplitude and Po).
The current analysis improperly mixes single-channel current amplitude and Po within the same figure, conflating distinct parameters. These quantities must be analyzed and presented separately, or the Po data should be removed entirely if not independently supported.
Insufficient event count.
Given the very limited number of observed openings, Po-based statistics are not meaningful. Please report only representative single-channel traces and corresponding amplitude histograms without attempting quantitative Po estimation.
Minor
Convert frequency (Hz) values to Po for consistency with earlier analyses, or remove frequency metrics altogether if Po analysis is omitted.
Figure 5 does not include Po or event frequency analysis, so we think there must be a misquotation of the figure. However, the Po issue has already been addressed before and alternative analysis have been proposed.
(6) Introduction
The introductory paragraph mentions the "five senses" as a framing concept. However, this statement lacks scientific grounding in the context of CSF-contacting neurons and chemosensory physiology. The traditional "five senses" classification is not an evidence-based neurophysiological framework and may be misleading to readers. I recommend removing or rephrasing this part, focusing instead on molecular and cellular mechanisms of sensory transduction (e.g., chemical, mechanical, and pH sensing) rather than on classical sensory categories.
Following the editor’s recommendation, we have removed this part.
The manuscript refers to PKD2L1 using the term TRPP2 in some parts of the introduction. This is incorrect, as PKD2L1 corresponds to TRPP3, not TRPP2. Please correct this nomenclature and ensure consistent use of "PKD2L1 (TRPP3)" throughout the entire manuscript to avoid confusion with the distinct PKD2/TRPP2 protein, which belongs to a different subfamily with separate physiological roles.
We thank the editor for pointing this out. As we mentioned in the responses to the “public reviews”, the literature is confusing, so we decided to remove from the manuscript any mention to TRPP channels.
(7) Discussion
The current Discussion reads largely as a descriptive summary of results and lacks conceptual depth. It does not effectively integrate the biophysical properties of PKD2L1 with its physiological role as a neuronal pH sensor, nor does it develop a broader interpretation relevant to CSF homeostasis or chemoreception.
Following the reviewers and editor’s recommendations, we have now added a new section in the Discussion untitled “PKD2L1 downstream signaling mechanisms”.
(8) Insufficient biophysical analysis
The discussion of channel gating and pH dependence is superficial and does not explore the energetic or structural mechanisms underlying proton sensitivity. The authors should analyze their data in the context of known PKD/TRPP family biophysics-for example, protonation sites, subunit composition, or gating kinetics-and explain how these confer bidirectional (acidic vs alkaline) sensitivity within physiological ranges.
In this work, we studied the pH sensitivity of PKD2L1 channels in the context of CSFcN sensory physiology. From a pure biophysical perspective, the pH sensitivity of PKD2L1 channels has been studied by multiple groups; however, it is still unknown how the gating of the channel responds to pH changes, although it can be proposed that some polar residues in the protein regulate the state of the pore. Likewise, the mechanism of the “off-response” is also unknown. To the best of our knowledge, there is only one article in which the authors have attempted to relate pH, PKD2L1 channel structure, and function. In this work (Su et al., Nature Communications 2018), the authors compare PKD2L1 channels with another pH-sensitive member of the TRP family, TRPML3, whose structures at pH 7.4 and 4.8 are known (Zhou et al., Nature Structural and Molecular Biology, 2017). We have rewritten some sentences of the Discussion in order to be more specific about the pH dependence of PKD2L1 channels and its proposed mechanisms.
(9) Weak physiological context
The manuscript does not adequately address how PKD2L1 functions as a true physiological pH sensor. The discussion should connect channel activity to realistic CSF pH fluctuations (6.8-7.6) and to relevant physiological or pathophysiological conditions (e.g., respiratory acidosis, neurogenic regulation of CSF composition). Without this, the relevance of large, artificial acidification (pH 3-3.5) remains unclear.
We have added a new section in the Discussion where we speculate on how PKD2L1 channels may be activated in physiological and pathophysiological conditions. However, we would like to insist here that the main goal of the photolysis experiments (which induce short and large acidifications) was to assess the spatial segregation of PKD2L1 channels. We now mention this point specifically and also speculate on the conditions that could eventually give rise to the “recovery” current.
(10) Over-interpretation of unsupported points
The paragraph describing voltage propagation from the ApPr to the soma/axon is speculative and unsupported by any data in the manuscript. Please delete this section entirely, including the citation to Orts-Del'immagine et al., unless new electrophysiological evidence is added.
We think this point (the propagation of signals originating from the ApPr to the soma) is important in the context of our work, so we have decided to make new experiments in order measure directly the degree of coupling between the 2 compartments. To do that we made simultaneous, current-clamp and voltage-clamp recordings from the ApPr and the soma. In these conditions we were able to measure experimentally and for the first time both the coupling coefficient and coupling conductance, which confirm that the propagation of voltage signals from the ApPr to the soma is extremely efficient. These new results are now described in a new subsection and in a new Figure 6.
(11) Clarify the role of "OFF currents."
The Discussion repeatedly refers to an "OFF response," but this phenomenon is not experimentally demonstrated for PKD2L1 alone. It likely represents pH-dependent recovery rather than an independent current. All discussion of "OFF currents" should be removed or reformulated accordingly.
Following the editor and reviewer’s comments, we have deleted the term “off response” and “off currents” from the ms and have replaced them with the term “recovery current”. We have also changed the discussion accordingly.
(12) Integration with ASICs and compartmental sensing
While the manuscript briefly mentions ASIC involvement, it does not articulate how PKD2L1- and ASIC-mediated signals might complement each other in different compartments (ApPr vs soma). The authors should discuss the potential division of labor between these sensors and how such compartmentalization enhances pH detection in CSFcNs.
Following the editor’s comments, we have rewritten the part of the subsection ‘the involvement of ASICs’ in the Discussion.
(12) Broadened physiological perspective
The Discussion should close by considering Ca2+ -dependent downstream pathways activated by ⁺ PKD2L1 and their implications for CSF flow regulation, neurosecretion, and central chemoreception. These translational aspects would substantially improve the impact and readability of the manuscript.
Done
Overall, the Discussion must evolve from a descriptive narrative to a mechanistically and physiologically integrative synthesis, highlighting why PKD2L1 is not merely present in the ApPr but is a key molecular transducer linking ionic microenvironment to neuronal excitability.
As it has been detailed above, we have performed several changes in the Discussion that follow the reviewer’s and editor’s recommendations.
