Author response:
Reviewer #1 (Public review):
Summary:
Urination requires precise coordination between the bladder and external urethral sphincter (EUS), while the neural substrates controlling this coordination remain poorly understood. In this study, Li et al. identify estrogen receptor 1-expressing neurons (ESR1+) in Barrington's nucleus as key regulators that faithfully initiate or suspend urination. Results from peripheral nerve lesions suggest that BarEsr1 neurons play independent roles in controlling bladder contraction and relaxation of the EUS. Finally, the authors performed region-specific retrograde tracing, claiming that distinct populations of BarEsr1 neurons target specific spinal nuclei involved in regulating the bladder and EUS, respectively.
Strength:
Overall, the work is of high quality. The authors integrate several cutting-edge technologies and sophisticated, thorough analyses, including opto-tagged single unit recordings, combined optogenetics, and urodynamics, particularly those following distinct peripheral nerve lesions.
Weakness:
(1) My major concern is the novelty of this study. Keller et al. 2018 have shown that BarEsr1 neurons are active during urination and play an essential role in relaxing the external urethral sphincter (EUS). Minimally, substantial content that merely confirms previous findings (e.g. Figures 1A-E; Figures 3A-E) should be move to the supplementary datasets.
Indeed, we are aware of and have carefully studied the literature of Keller et al. Our manuscript here presents novel experiments beyond the scopes of that paper. Thanks to this comment, we will substantially revise our manuscript to enhance the visibility of novel data while keeping the agreeing data in the supplementary.
(2) I also have concerns regarding the results showing that the inactivation of BarEsr1 neurons led to the cessation of EUS muscle firing (Figures 2G and S5C). As shown in the cartoon illustration of Figure 8, spinal projections of BarEsr1 neurons contact interneurons (presumably inhibitory) that innervate motor neurons, which in turn excite the EUS. I would therefore expect that the inactivation of BarEsr1 should shift the EUS firing pattern from phasic (as relaxation) to tonic (removal of relaxation), rather than stopping their firing entirely. Could the authors comment on this and provide potential reasons or mechanisms for this finding?
We agree with this point. We meant that the EUS’ phasic bursting pattern was rapidly stopped upon BarEsr1 photoinhibition, but not all the firing stopped instantaneously. According to the previous studies (Chang et al., 2007, de Groat, 2009, de Groat and Yoshimura, 2015, Kadekawa et al., 2016), the voiding physiology of rodents is probably different from that of humans, such that for rodents the urine is step-wise pumped out in the gap time between multiple consecutive EUS phasic bursting epochs, and for humans the urine is continuously pumped out once the EUS firing is almost fully inhibition during a period of time. Namely, for mice, the EUS display sustained tonic activity following phasic bursting, while, in contrast, for humans the EUS keeps tonic firing until the moment of voiding onset (complete inhibition, muscle relaxed). Despite the prominent differences in the basic physiological properties, our assumption is that the logic of circuits from the brainstem to the urethra in this pathway is evolutionally conserved for both species; thus the logic of brainstem coordination of voiding could also be the same for both species, which is the main interest of our study (of using an animal model to address concerns of human health). Thus, to interpret our data for a broader audience we made a simplified and inaccurate expression. We apologize for the inaccuracy and we will correct our previous inaccurate description in the revised manuscript.
(3) Current evidence is insufficient to support the claim that the majority of BarEsr1 neurons innervate the SPN but not DGC. The current spinal images are uninformative, as the fluorescence reflects the distribution of Esr1- or Crh-expressing neurons in the spinal cord, along with descending BarEsr1 or BarCrh axons. Given the close anatomical proximity of these two nuclei, a more thorough histological analysis is required to demonstrate that the spinal injections were accurately confined to either the SPN or the DGC.
We agree that current evidence is insufficient to support the current claim. To address this concern and strengthen our claim, we will repeat the retrograde viral tracing experiments, combined with CTB647 injections to label the injection site, to validate specific targeting of SPN or DGC populations. We will also add higher-magnification imaging to distinguish BarESR1 axonal projections targeting SPN versus DGC. Results from these ongoing experiments will be incorporated into the revised manuscript.
Reviewer #2 (Public review):
Summary:
The authors have performed a rigorous study to assess the role of ESR1+ neurons in the PMC to control the coordination of bladder and sphincter muscles during urination. This is an important extension of previous work defining the role of these brainstem neurons, and convincingly adds to the understanding of their role as master regulators of urination. This is a thorough, well-done study that clarifies how the Pontine micturition center coordinates different muscle groups for efficient urination, but there are some questions and considerations that remain.
Strengths:
These data are thorough and convincing in showing that ESR1+PMC neurons exert coordinated control over both the bladder and sphincter activity, which is essential for efficient urination. The anatomical distinctions in pelvic versus pudendal control are clear, and it's an advance to understand how this coordination occurs. This work offers a clearer picture of how micturition is driven.
Weaknesses:
The dynamics of how this population of ESR1+ neurons is engaged in natural urination events remains unclear. Not all ESR1+neurons are always engaged, and it is not measured whether this is simply variation in population activity, or if more neurons are engaged during more intense starting bladder pressures, for instance. In particular, the response dynamics of single and doubly-projecting neurons are not defined. Additionally, the model for how these neurons coordinate with CRH+ neuron activity in the PMC is not addressed, although these cell types seem to be engaged at the same time. Lastly, it would be interesting to know how sensory input can likely modulate the activity of these neurons, but this is perhaps a future direction.
In response to the reviewer’s comments, we will attempt perform the following revisions for this round:
(1) Engagement of ESR1+ neurons in natural urination events:
We agree that probably not all ESR1+ neurons are consistently engaged during urination. To address this, we will perform a detailed analysis of the opto-tagged single unit recordings data.
(2) Response dynamics of single- and doubly-projecting neurons:
(a) We will use retrograde labelling combined with Ca2+ photometry recordings to differentiate the response dynamics of SPN- and DGC-projecting neurons during urination.
(b) We will perform functional validations to assess the specific roles of single- and doubly-projecting neurons in coordinating bladder and EUS activity.
(3) Coordination with CRH+ neurons in the PMC:
We appreciate the suggestion to include CRH+ neurons in our model. We will expand our model to incorporate CRH+ neurons and their potential interactions with ESR1+ neurons.
(4) Sensory modulation of ESR1+ neurons:
The reviewer raises an excellent point regarding sensory input modulation of ESR1+ neuron activity. Although this is beyond the scope of our current study, we recognize its importance and propose to include this as a future direction.
Reviewer #3 (Public review):
Summary:
The paper by Li et al explored the role of Estrogen receptor 1 (Esr1) expressing neurons in the pontine micturition center (PMC), a brainstem region also known as Barrington's nucleus (Hou et al 2016, Keller et al 2018). First, the author conducted bulk Ca2+ imaging/unit recording from PMCESR1 to investigate the correlations of PMCESR1 neural activity to voiding behavior in conscious mice and bladder pressure/external urethral muscle activity in urethane anesthetized mice. Next, the authors conducted optogenetics inactivation/activation of PMCESR1 to confirm the contribution to the voiding behavior also conducted peripheral nerve transection together with optogenetics activation to confirm the independent control of bladder pressure and urethral sphincter muscle.
Weaknesses:
(1) The study demonstrates that pelvic nerve transection reduces urinary volume triggered by PMCESR1+ cell photoactivation in freely moving mice. Could the role of pudendal nerve transection also be examined in awake mice to provide a more comprehensive understanding of neural involvement?
Thank you for the suggestion, the pudendal nerve transection in awake mice is indeed a challenging experiment that has been missed. We will try it for the revision.
(2) While the paper primarily focuses on PMCESR1+ cells in bladder-sphincter coordination, the analysis of PMCESR1+-DGC/SPN neural circuits - given their distinct anatomical projections in the sacral spinal cord - feels underexplored. How do these circuits influence bladder and sphincter function when activated or inhibited? Also, do you have any tracing data to confirm whether bladder-sphincter innervation comes from distinct spinal nuclei?
Thank you for this great comment. The projection-specific neuronal function analysis is, as also suggested by Reviewer 2 in a similar comment (#8), missing in our first submission. These are so challenging experiments that we have missed in the first round of tests, but we decide to pursuit this goal again. Namely, we will perform photometry recordings of PMC neurons projecting to the DGC/SPN during measuring bladder pressure and urethral sphincter EMG activity. Additionally, while our study does not include direct tracing data to confirm distinct spinal nuclei for bladder and sphincter innervation, this has been well-documented in classic literature (Yao et al., 2018, Karnup and De Groat, 2020, Karnup, 2021). Specifically, anatomical studies have shown that SPN primarily innervates the bladder, while the DGC is associated with the innervation of the urethral sphincter. We will cite these references to provide context and support for our interpretations.
(3) Although the paper successfully identifies the physiological role of PMCESR1+ cells in bladder-sphincter coordination, the study falls short in examining the electrophysiological properties of PMCESR1+-DGC/SPN cells. A deeper investigation here would strengthen the findings.
While our study primarily focuses on the functional role of PMCESR1+ neurons in bladder-sphincter coordination, we acknowledge that understanding their intrinsic electrophysiological characteristics could further strengthen our findings. However, this aspect falls beyond the scope of the current study. Nevertheless, we recognize the significance of this direction and are excited to pursue it in future research. We appreciate the reviewer’s suggestion, as it highlights an important avenue for expanding upon our current findings.
(4) The parameters for photoactivation (blue light pulses delivered at 25 Hz for 15 ms, every 30 s) and photoinhibition (pulses at 50 Hz for 20 ms) vary. What drove the selection of these specific parameters? Moreover, for photoactivation experiments, the change in pressure (ΔP = P5 sec - P0 sec) is calculated differently from photoinhibition (Δpressure = Ppeak - Pmin). Can you clarify the reasoning behind these differing approaches?
We sincerely thank the reviewer for raising these important points and for the opportunity to clarify our experimental design and data analysis methods.
Photoactivation versus photoinhibition parameters: The differences in photoactivation (25 Hz, 15 ms pulses) and photoinhibition (50 Hz, 20 ms pulses) protocols are based on the distinct physiological and technical requirements for activating versus inhibiting PMCESR1+ neurons. For photoactivation, 25 Hz stimulation aligns with the natural firing patterns of central neurons, allowing for intermittent activation without exceeding the neuronal refractory period. The shorter pulse duration (15 ms) minimizes phototoxicity and avoids overstimulation, as performed in previous studies (Keller et al., 2018). In contrast, photoinhibition requires sustained suppression of neuronal activity, achieved through higher frequencies (50 Hz) and longer pulses (20 ms) to ensure continuous coverage of neuronal activity.
Calculation of pressure changes (ΔP) for photoactivation and photoinhibition: The differing methods for calculating pressure changes reflect the distinct physiological effects we aimed to capture. In photoactivation experiments (ΔP = P5 sec - P0 sec), the pressures before (P0 sec) and 5 seconds after (P5 sec) light delivery were compared to capture the immediate effect of light activation on bladder pressure, focusing on the onset and early dynamics of activation. In contrast, photoinhibition experiments assessed the immediate impact of light-induced suppression on bladder pressure during an ongoing voiding event. Here, Δpressure was calculated as Ppeak – Pmin to measure the rapid drop in pressure directly attributable to neuronal inhibition.
We will expand these details in the methods section of the revised manuscript to provide greater transparency.
(5) The discussion could further emphasize how PMCESR1+ cells coordinate bladder contraction and sphincter relaxation to control urination, highlighting their central role in the initiation and suspension of this process.
We fully agree with this point. Additionally, in response to your and other reviewers’ suggestions, we are preparing a new round of experiments with projection-specific recording, and thus our discussion and conclusion will also be updated according to the newly obtained data.
(6) In Figure 8, The authors analyze the temporal sequence of bladder pressure and EUS bursting during natural voiding and PMC activation-induced voiding. It would be acceptable to consider the existence of a lower spinal reflex circuit, however, the interpretation of the data contains speculation. Bladder pressure measurement is hard to say reflecting efferent pelvic nerve activity in real time. (As a biological system, bladder contraction is mediated by smooth muscle, and does not reflect real-time efferent pelvic nerve activity. As an experimental set-up, bladder pressure measurement has some delays to reflect bladder pressure because of tubing, but EUS bursting has no delay.) Especially for the inactivation experiment, these factors would contribute to the interpretation of data. This reviewer recommends a rewrite of the section considering these limitations. Most of the section is suitable for the results.
Thank you for mentioning the possibility of bladder pressure measurement delay. We would prefer to perform a physical control test to quantify how much delay this measurement is under our experimental conditions. We will use a small ballon to mimic the bladder and use two identical pressure sensors, one with a very short tube inserted into the ballon and one with an extended tube same as in our animal experiments. We will then mimic both contraction initiation and halting, and quantify the delay between the two sensors.
References
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de Groat WC. 2009. Integrative control of the lower urinary tract: preclinical perspective. British Journal of Pharmacology 147. DOI: 10.1038/sj.bjp.0706604
de Groat WC, Yoshimura N. 2015. Anatomy and physiology of the lower urinary tract. Handb Clin Neurol 130: 61-108. DOI: 10.1016/B978-0-444-63247-0.00005-5
Kadekawa K, Yoshimura N, Majima T, Wada N, Shimizu T, Birder LA, Kanai AJ, de Groat WC, Sugaya K, Yoshiyama M. 2016. Characterization of bladder and external urethral activity in mice with or without spinal cord injury—a comparison study with rats. American Journal of Physiology-Regulatory, Integrative and Comparative Physiology 310: R752-R758. DOI: 10.1152/ajpregu.00450.2015
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Keller JA, Chen J, Simpson S, Wang EH-J, Lilascharoen V, George O, Lim BK, Stowers L. 2018. Voluntary urination control by brainstem neurons that relax the urethral sphincter. Nature Neuroscience 21: 1229-1238. DOI: 10.1038/s41593-018-0204-3
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