Author response:
The following is the authors’ response to the previous reviews.
eLife assessment
Chang et al. provide glutamate co-expression profiles in the central noradrenergic system and test the requirement of Vglut2-based glutamatergic release in respiratory and metabolic activity under physiologically relevant gas challenges. Their experiments provide compelling evidence that conditional deletion of Vglut2 in noradrenergic neurons does not impact steadystate breathing or metabolic activity in room air, hypercapnia, or hypoxia. This study provides an important contribution to our understanding of how noradrenergic neurons regulate respiratory homeostasis in conscious adult mice.
Public Reviews:
Reviewer #1 (Public Review):
Summary:
Chang et al. provide glutamate co-expression profiles in the central noradrenergic system and test the requirement of Vglut2-based glutamatergic release in respiratory and metabolic activity under physiologically relevant gas challenges. Their experiments show that conditional deletion of Vglut2 in NA neurons does not impact steady-state breathing or metabolic activity in room air, hypercapnia, or hypoxia. Their observations challenge the importance of glutamatergic signaling from Vglut2 expressing NA neurons in normal respiratory homeostasis in conscious adult mice.
Strengths:
The comprehensive Vglut1, Vglut2, and Vglut3 co-expression profiles in the central noradrenergic system and the combined measurements of breathing and oxygen consumption are two major strengths of this study. Observations from these experiments provide previously undescribed insights into (1) expression patterns for subtypes of the vesicular glutamate transporter protein in the noradrenergic system and (2) the dispensable nature of Vglut2-dependent glutamate signaling from noradrenergic neurons to breathing responses to physiologically relevant gas challenges in adult conscious mice.
Weaknesses:
Although the cellular expression profiles for the vesicular glutamate transporters are provided, the study fails to document that glutamatergic-based signaling originating from noradrenergic neurons is evident at the cellular level under normal, hypoxic, and/or hypercapnic conditions. This limits the reader's understanding of why conditional Vglut2 knockdown is dispensable for breathing under the conditions tested.
We thank the reviewers for their positive evaluation of our work. First, we would like to highlight that multiple studies have provided anatomical evidence of innervation of multiple cardio-respiratory nuclei by Vglut2+ noradrenergic fibers. Thus, the anatomical substrates are present for noradrenergic based Vglut2 signaling to either play a direct role in breathing control or, upon perturbation, to indirectly affect breathing through disrupted metabolic or cardiovascular control. We have included supplemental table 1 that summarizes central noradrenergic Vglut2+ innervations of respiratory and autonomic nuclei. Additionally, Ultrastructural evidence shows asymmetric synaptic contacts assuming glutamatergic transmission between C1 neurons and LC, A1, A2 and the dorsal motor nucleus of the vagus (DMV) (Milner et al., 1989; Abbott et al., 2012; Holloway et al., 2013; DePuy et al., 2013).
Functionally, electrophysiological evidence showed that photostimulating C1 neurons activate LC, A1, A2 noradrenergic neurons monosynaptically by releasing glutamate (Holloway et al., 2013; DePuy et al., 2013) and optogenetic stimulation of LC neurons excite the downstream parabrachial nucleus (PBN) neurons by releasing glutamate. Thus, at least the glutamatergic signaling from C1 and LC noradrenergic neurons (two noradrenergic nuclei that have been shown to play a role in breathing control) is evident at the cellular level under normal conditions. Other evidence, highlighted in our manuscript, is more circumstantial.
Reviewer #2 (Public Review):
The authors characterized the recombinase-based cumulative fate maps for vesicular glutamate transporters (Vglut1, Vglut2 and Vglut3) expression and compared those maps to their real-time expression profiles in central NA neurons by RNA in situ hybridization in adult mice. Authors have revealed a new and intriguing expression pattern for Vglut2, along with an entirely uncharted co-expression domain for Vglut3 within central noradrenergic neurons. Interestingly, and in contrast to previous studies, the authors demonstrated that glutamatergic signaling in central noradrenergic neurons does not exert any influence on breathing and metabolic control either under normoxic/normocapnic conditions or after chemoreflex stimulation. Also, they showed for the first-time the Vglut3-expressing NA population in C2/A2 nuclei. In addition, they were also able to demonstrate Vglut2 expression in anterior NA populations, such as LC neurons, by using more refined techniques, unlike previous studies.
A major strength of the study is the use of a set of techniques to investigate the participation of NA-based glutamatergic signaling in breathing and metabolic control. The authors provided a full characterization of the recombinase-based cumulative fate maps for Vglut transporters. They performed real-time mRNA expression of Vglut transporters in central NA neurons of adult mice. Further, they evaluated the effect of knocking down Vglut2 expression in NA neurons using a DBH-Cre; Vglut2cKO mice on breathing and control in unanesthetized mice. Finally, they injected the AAV virus containing Cre-dependent Td tomato into LC of v-Glut2 Cre mice to verify the VGlut2 expression in LC-NA neurons. A very positive aspect of the article is that the authors combined ventilation with metabolic measurements. This integration holds particular significance, especially when delving into the exploration of respiratory chemosensitivity. Furthermore, the sample size of the experiments is excellent.
Despite the clear strengths of the paper, some weaknesses exist. It is not clear in the manuscript if the experiments were performed in males and females and if the data were combined. I believe that the study would have benefited from a more comprehensive analysis exploring the sex specific differences. The reason I think this is particularly relevant is the developmental disorders mentioned by the authors, such as SIDS and Rett syndrome, which could potentially arise from disruptions in central noradrenergic (NA) function, exhibit varying degrees of sex predominance. Moreover, some of the noradrenergic cell groups are sexually dimorphic. For instance, female Wistar rats exhibit a larger LC size and more LC-NA neurons than male subjects (Pinos et al., 2001; Garcia-Falgueras et al., 2005). More recently, a detailed transcriptional profiling investigation has unveiled the identities of over 3,000 genes in the LC. This revelation has highlighted significant sexual dimorphisms, with more than 100 genes exhibiting differential expression within LC-NA neurons at the transcript level. Furthermore, this investigation has convincingly showcased that these distinct gene expression patterns have the capacity to elicit disparate behavioral responses between sexes (Mulvey et al., 2018). Therefore, the authors should compare the fate maps, Vglut transporters in males and females, at least considering LC-NA neurons. Even in the absence of identified sex differences, this information retains significant importance.
All experiments contained both males and females as described in the original submission. In our analysis of breathing and metabolism, sex was included in the analysis and no significant phenotypic difference was observed. For the fate map and in situ experiments, we did not see obvious differences in the expression patterns in the three glutamate transporters between females and males, though the group size is small. Though all the anatomical and phenotypic data in this manuscript are presented as combined graphs, we have differentially labeled our data points by sex. The reviewer does raise important questions regarding possible sexual dimorphisms in the central noradrenergic system and whether such dimorphisms may extend to glutamate transporter co-expression. Our thorough interrogation of respiratory-metabolic parameters fails to reveal any sex specific differences in control or experimental mice. Thus, it is unclear if any of the previously described and cited dimorphisms are functionally relevant in this setting. Given the large differences in the real time expression and cumulative fate maps of Vglut2, a worthwhile interrogation of differential glutamate transporter expression would be best served by longitudinal studies with large group sizes across age as it is not clear what underlies the dynamic VGlut2 expression changes. Such changes may at times be greater in males and other times in females, driven by experience or physiological challenges etc., but resulting in averaged cumulative fatemaps that are similar between sexes. Such a longitudinal quantitative study of real-time and fatemapped cell populations across the central NA system would be of a scale that is beyond the scope of this report, especially when no phenotypic changes have been observed in our respiratory data.
An important point well raised by the authors is that although suggestive, these experiments do not definitively rule out that NA-Vglut2 based glutamatergic signaling has a role in breathing control. Subsequent experiments will be necessary to validate this hypothesis.
As noted, we discuss that we only address requirement, not sufficiency, of NA Vglut2 in breathing. Functional sufficiency experiments usually involve increasing the relevant output. However, these experiments can lead to non-specific, pleiotropic effects that would be difficult to disambiguate, even if done with high cellular specificity. Viral or genetic overexpression of Vglut2 in NA neurons may be a feasible approach. Conditional ablation of TH or DBH with concurrent chemo or optogenetic stimulation may also be informative. These approaches would require significant investments in mouse model generation and suffer additional experimental limitations.
An improvement could be made in terms of measuring body temperature. Opting for implanted sensors over rectal probes would circumvent the need to open the chamber, thereby preventing alterations in gas composition during respiratory measurements. Further, what happens to body temperature phenotype in these animals under different gas exposures? These data should be included in the Tables.
While surgical implantation of sensors would provide a more direct assessment of temperature, it requires components that were not available at the time of the study and addresses a question (temperature changes during a time course of gas exposure) that go beyond the scope of the current work focused on respiratory response. As we have done for prior experiments (Martinez et al., 2019; Ray et al., 2011), the body temperature was measured immediately before and after measuring breathing only. Our flow through system using inline gas sensors (AEI P-61B CO2 sensor and AEI N-22M O2 sensor) ensure that gas challenges were constant and consistent across all measurements. Any disruption in gas composition would have been noted by our software analysis system, Breathe Easy, and the data rejected. We did not observe any such perturbations.
Is it plausible that another neurotransmitter within NA neurons might be released in higher amounts in DBH-Cre; Vglut2 cKO mice to compensate for the deficiency in glutamate and prevent changes in ventilation?
We agree that compensation is always a possibility at the synaptic, cellular, and circuit levels that may involve a variety of transcriptional, translational, cellular, and circuit mechanisms (i.e., synaptic strength). This could be interrogated by combining multiple conditional alleles and recombinase drivers for various transmitters and receptors, but would, in our experience, take multiple years for the requisite breeding to be completed.
Continuing along the same line of inquiry is there a possibility that Vglut2 cKO from NA neurons not only eliminates glutamate release but also reduces NA release? A similar mechanism was previously found in VGLUT2 cKO from DA neurons in previous studies (Alsio et al., 2011; Fortin et al., 2012; Hnasko et al., 2010). Additionally, does glutamate play a role in the vesicular loading of NA? Therefore, could the lack of effect on breathing be explained by the lack of noradrenaline and not glutamate?
These are all excellent points, but prior studies suggest that reductions in NA signaling would itself have an apparent effect (Zanella et al., 2006; Kuo et al., 2016). Although several studies showed that LC and C1 NA neurons co-release noradrenaline and glutamate, no direct evidence yet makes clear that glutamate facilitates NA release or vice versa. However, it would be of great interest to test if reduced or lack of NA compensated for loss of glutamate in the future. We do fully acknowledge that compensation in the manuscript that any number of compensatory events could be at play in these findings.
Reviewer #3 (Public Review):
Summary:
The authors, Y Chang and colleagues, have performed elegant studies in transgenic mouse models that were designed to examine glutamatergic transmission in noradrenergic neurons, with a focus on respiratory regulation. They generated 3 different transgenic lines, in which a red fluorophore was expressed in dopamine-B-hydroxylase (DBH; noradrenergic and adrenergic neurons) neurons that did not express a vesicular glutamate transporter (Vglut) and a green fluorophore in DBH neurons that did express one of either Vglut1, Vglut2 or Vglut3.
Further experiments generated a transgenic mouse with knockout of Vglut2 in DBH neurons. The authors used plethysmography to measure respiratory parameters in conscious, unrestrained mice in response to various challenges.
Strengths:
The distribution of the Vglut expression is broadly in agreement with other studies, but with the addition of some novel Vglut3 expression. Validation of the transgenic results, using in situ hybridization histochemistry to examine mRNA expression, revealed potential modulation of Vglut2 expression during phases of development. This dataset is comprehensive, wellpresented and very useful.
In the physiological studies the authors observed that neither baseline respiratory parameters, nor respiratory responses to hypercapnea (5, 7, 10% CO2) or hypoxia (10% O2) were different between knockout mice and littermate controls. The studies are well-designed and comprehensive. They provide observations that are supportive of previous reports using similar methodology.
Weaknesses:
In relation to the expression of Vglut2, the authors conclude that modulation of expression occurs, such that in adulthood there are differences in expression patterns in some (nor)adrenergic cell groups. Altered sensitivity is provided as an explanation for different results between studies examining mRNA expression. These are likely explanations; however, the conclusion would really be definitive with inclusion of a conditional cre expressing mouse. Given the effort taken to generate this dataset, it seems to me that taking that extra step would be of value for the overall understanding of glutamatergic expression in these catecholaminergic neurons
The seemingly dynamic Vglut2 expression pattern across the NA system is intriguing. As noted in our comments to reviewer 2, a robust age dependent interrogation would require a large magnitude study. The reviewer correctly points out that a temporally controlled recombinase fate mapping experiment would offer greater insight into the dynamic expression of Vglut2. We strongly agree with that idea and did work to develop a Vglut2-CreER targeted allele that, despite our many other successes in mouse genetic engineering (Lusk et al., 2022; Sun and Ray, 2016), did not succeed on the first attempt. We aim to complete the line in the near future so that we may better understand the Vglut2 expression pattern in central noradrenergic neurons in a time-specific manner and sex specific manner.
The respiratory physiology is very convincing and provides clear support for the view that Vglut2 is not required for modulation of the respiratory parameters measured and the reflex responses tested. It is stated that this is surprising. However, comparison with the data from Abbott et al., Eur J Neurosci (2014) in which the same transgenic approach was used, shows that they also observed no change in baseline breathing frequency. Differences were observed with strong, coordinated optogenetic stimulation, but, as discussed in this manuscript, it is not clear what physiological function this is relevant to. It just shows that some C1 neurons can use glutamate as a signaling molecule. Further, Holloway et al., Eur J Neurosci (2015), using the same transgenic mouse approach, showed that the respiratory response to optogenetic activation of Phox2 expressing neurons is not altered in DBH-Vglut2 KO mice. The conclusion seems to be that some C1 neuron effects are reliant upon glutamatergic transmission (C1DMV for example), and some not.
We agree that activation of C1 neurons may be sufficient to modulate breathing when artificially stimulated and that such stimulation relies on glutamatergic transmission for its effect. This is why we find our results surprising and important in clarifying for the field that glutamatergic signaling in noradrenergic cells is dispensable for breathing and hypoxic and hypercapnic responses under physiological conditions.
Further contrast is made in this manuscript to the work of Malheiros-Lima and colleagues (eLife 2020) who showed that the activation of abdominal expiratory nerve activity in response to peripheral chemoreceptor activation with cyanide was dependent upon C1 neurons and could be attenuated by blockade of glutamate receptors in the pFRG - i.e. the supposition that glutamate release from C1 neurons was responsible for the function. However, it is interesting to observe that diaphragm EMG responses to hypercapnia (10% CO2) or cyanide, and the expiratory activation to hypercapnia, were not affected by the glutamate receptor blockade. Thus, a very specific response is affected and one that was not measured in the current study.
As we mention above, we do not dispute that glutamate signaling can be manipulated to create a response in non-physiological conditions – we suggest that framing the interpretation around the glutamatergic role in a model that better matches physiological conditions should inform our interpretation. Furthermore, we do include an examination of expiratory flow – which was not impacted by loss of glutamatergic activity in NA neurons – which would be likely to have been impacted if abdominal expiratory nerve activity was modified.
These previous published observations are consistent with the current study which provides a more comprehensive analysis of the role of glutamatergic contributions respiratory physiology. A more nuanced discussion of the data and acknowledgement of the differences, which are not actually at odds, would improve the paper and place the information within a more comprehensive model.
Thank you for the comments. As noted in the original and extended discussion, we respectfully disagree with the perspective that our results align with prior results.
Recommendations for the authors:
The three reviewers believe this is an important study. They have numerous suggestions for improvement of the manuscript (outlined below), but no new experiments are required. The Editor requests some nomenclature changes as indicated in attachment 1.
Reviewer #1 (Recommendations For The Authors):
Abstract/Introduction: Although the need for this study is obvious, it is important that the authors explicitly communicate their working hypothesis < before the start of the work> to the reader. In the current form, it is unclear whether the authors aimed to test the hypothesis that glutamatergic signaling from noradrenergic neurons is important to breathing or whether to test the hypothesis that glutamatergic signaling from noradrenergic neurons is not important to breathing. If it is the latter-it is not important-then the study (related to the breathing measurements) is poorly justified and designed, as additional orthogonal approaches (e.g., actual measurements of glutamatergic signaling at the cellular level) are almost requisite. If the authors' hypothesis was originally based on existing literature suggesting that glutamatergic signaling from noradrenergic neurons is important to breathing, then the experimental design appropriate.
Thank you for the suggestion. The working hypothesis has been added in the abstract (line 2425) and the introduction (line 92-94)), making clear that we initially hypothesized that glutamatergic signaling from noradrenergic neurons is important in breathing.
Results: While the steady state measurements for breathing metrics are clearly important in defining how glutamatergic signaling may contribute to be pulmonary function, the role of glutamatergic signaling may have a greater role in the dynamics of patterns (i.e., regularity of the breathing rhythms) such traits can be described using SD1 and SD2 from Poincare maps, and/or entropy measurements. Such an analysis should be performed.
Thank you for the suggestion. The dynamic patterns of respiratory rate (Vf), tidal volume (VT), minute ventilation (VE), inspiratory duration (TI), expiratory duration (TE), breath cycle duration (TTOT), inspiratory flow rate (VT/TI), expiratory flow rate (VT/TE) have been shown as Poincaré plots and quantified and tested using the SD1 and SD2 statistics in the supplemental figures of Figure 4-7.
Results: Analyses of Inspiratory time (Ti) and flow rate (i.e., Tidal Volume / Ti) should be assessed and included.
Thank you for the suggestion. Inspiratory duration (Ti), expiratory duration (TE), breath cycle duration (TTOT), inspiratory flow rate (VT/Ti), and expiratory flow rate (VT/TE) have been included in the Figures 4-7.
Results/Methods: If similar analytical approaches were used in the current study as to that in Lusk et al. 2022, it appears that data was discontinuously sampled, rejecting periods of movement and only including periods of quiescent breathing. Were the periods of quiescent breathing different? Information should be provided to describe the total sampling duration included.
For room air, the entire gas condition was used for data analysis. For hypercapnia (5% CO2, 7% CO2, 10% CO2), only the last 5 minutes of the gas challenge period was used for data analysis. For hypoxia (10% O2), we analyzed the breathing trace of three 5-minute epochs following initiation of the gas exposure separately, e.g., epoch 1 = 5-10min, epoch 2 = 10-15min, and epoch 3 = 15-20min. All breaths included as quiescent breathing were analyzed in the aggregate for each group and experimental condition, we did not compare individual periods of quiescent breathing within or across an animal(s)/group(s)/experimental condition(s). We have added the details in the Materials and Methods (line 637-642).
Results: As mice were conscious in this study, were sniff periods (transient periods of fast breathing, i.e.,>8Hz) included in the analysis?
No, only regular quiescent breathing periods were included in the analysis.
Discussion: The authors need to discuss the limitations of their findings.
- How should the reader interpret the findings? Concluding that glutamatergic signaling is dispensable implies that it occurs in room air, hypoxia, and hypercapnia.
We have edited our discussion for clarity to highlight our conclusions that Vglut2-based glutamatergic signaling from noradrenergic neurons is ultimately dispensable for baseline breathing and hypercapnia and hypoxic chemoreflex in unanesthetized and unrestrained mice.
- Assuming that glutamatergic signaling is active during the conditions tested, then the authors should discuss what may be the potential compensations.
We have provided additional discussion surrounding potential compensatory events that may have taken place and could result in the unchanged phenotype in the experimental group.
- The authors need to discuss how age and state of consciousness may play a role in their finds. The current discussion gives the impression that their findings are broadly applicable in all cases, but the lack of differences in this study may not hold true under different conditions.
The study was done in adult (6–8-week-old) unanesthetized and unrestrained mice. In the discussion (line 472-474), we highlight that in our unpublished results, loss of NA-expressed Vglut2 does not change the survival curve in P7 neonate mice undergoing repeated bouts of autoresuscitation until death. Thus, we believed that Vglut2-based glutamatergic signaling in central NA neurons is dispensable for baseline breathing and the hypercapnic and hypoxic chemoreflexes in unanesthetized and unrestrained mice across different ages. Otherwise, we do not imply that we have interrogated any other aspects of breathing in our discussion.
Methods: Further description of the analysis window for the respiratory metrics should be provided. Were breath values for each condition taken throughout the entire condition? This is particularly important for hypoxia, where the stereotypical respiratory response is biphasic.
For room air, the entire gas condition was used for data analysis. For hypercapnia (5% CO2, 7% CO2, 10% CO2), only the last 5min of the gas challenge period was used for data analysis. For hypoxia (10% O2), we analyzed the breathing trace of three 5min time periods separately including 5-10min, 10-15min, and 15-20min during the hypoxic challenge as noted in our original manuscript, we graph and assess three 5min epochs during hypoxic exposure to capture the dynamic nature of the hypoxic ventilatory response. We have added the details in the Materials and Methods (line 637-642).
Methods: How was consciousness determined?
The conscious mice mentioned in the manuscript refer to the mice without anesthesia. We have replaced “awake” and “conscious” with “unanesthetized” in the text.
Reviewer #2 (Recommendations For The Authors):
Since no EEG/EMG recording was performed it would be more appropriate to remove "awake" and "conscious" throughout the manuscript and include the term "unanesthetized".
Thank you for the suggestion. “Awake” and “conscious” have been replaced by “unanesthetized” in the text.
Line 545: Why 32C? Isn't this temperature too high for animals?
30-32°C is the thermoneutral zone for mice. It is the range of ambient temperature where mice can maintain a stable core temperature with their minimal metabolic rate (Gordon, 1985). Whole-body plethysmography uses the barometric technique to detect pressure oscillations caused by changes in temperature and humidity with each breathing act when an animal sits in a sealed chamber (Mortola et al., 2013). Thus, maintaining the chamber temperature near the thermoneutral zone during the plethysmography assay is required to maintain constancy in respiratory and metabolic parameters from trial to trial as well as to maintain linearity of ventilatory pressure changes due to humidification, rarefaction, and thermal expansion and contraction during inspiration and expiration (Ray et al., 2011). The chamber temperature that has been used for adult plethysmography has been set across a range 30-34°C (Hodges et al., 2008; Ray et al., 2011; Hennessy et al., 2017). We use 32°C in this manuscript which is consistent with previously published literature from other groups and our own work (Sun et al., 2017; Lusk et al., 2022).
I would include the units of the physiological variables in the tables.
Thank you for the suggestion. The units of the physiological variables have been added in all the tables.
Reviewer #3 (Recommendations For The Authors):
Why is the C3 group not considered in this study?
The C3 adrenergic group, best characterized in rat, is only seen in rodents but not in many other species including primates (including human) (Kitahama et al., 1994). Thus, the C3 group is not the focus of this study where we aim to discuss if glutamate derived from noradrenergic neurons could be the potential therapeutic target of human respiratory disorders. The C3 adrenergic group is typically described as a population containing only about 30 neurons. We have added the fate map data and the adult expression pattern for the three vesicular glutamate transporters for the C3 group in the figure 1 and 2 supplements for reference.
Sub CD/CV does not appear to be defined in the manuscript.
Thank you for the point. The definition of sub CD/CV has been added in the text (line 126).
The data on line 131-133 is interesting but could be described more effectively and clearly.
Thank you for the suggestion. The text has been modified accordingly.
The end of the paragraph at lines 140 onwards is rather repeated in the paragraph that starts at line 146.
The repeated text has been removed accordingly.
Whilst anterior and posterior are correct anatomical terms, for a quadraped, rostral and caudal are more widely used - particularly in the brainstem field. Is there a particular reason for using anterior/posterior?
We followed the anatomical terminations in the Robertson et al. (2013) where they used anterior/posterior to describe C2/A2 and C1/A1.
On the protocol lines include in Figure 4-7 it would be worth adding the test day. This seems a little strange. Why wait up to one week after the habituation to perform the stimulation. How many mice were left for each day between habituation and experimentation, and does this timing affect responses? Do mice forget the habituation after a period?
Thank you for the point. We have added the test day for plethysmography in figures 4-7. After the 5 days of habituation, we began the plethysmography recordings on the sixth day. A maximum of 6 mice can be assayed for plethysmography per day due to the limited number of barometric flow through plethysmography and metabolic measurement systems we have. Thus, all animals were finished with plethysmography “within” one week of the last day of habituation. This protocol is consistent with our previous published work (Martinez et al., 2019; Lusk et al., 2022; Lusk et al., 2023). For the experiments in this manuscript, mice were assayed within 3 days after habituation. As noted in our methods and figures, each mouse is given as much as 40 mins to acclimate to the chamber (determined by directly observed quiet breathing) before data acquisition. We have no reason or evidence that indicates testing order and thus timing was a factor. The detailed explanation for the plethysmography protocol has been added in the material and methods section (line 606-625).
Please state clearly that each mouse is only exposed to one gas mixture (what I interpret is the case), or could one mouse be exposed to several different stimuli?
Each mouse is only exposed to one gas challenge (5% CO2, 7% CO2, 10% CO2, or 10% O2) in a testing period. Each testing period for an individual mouse was separated by 24hs to allow for a full recovery. The protocol is to put the mouse under room air for 45mins, switch to one gas challenge for 20mins, and switch back to room air for 20mins.
With apologies if I missed this, but did each of the respiratory stimuli produce a statistically significant response in the control mice? For example, the response to 10%O2?
Yes, each respiratory stimuli including 5/7/10% CO2 and 10% O2 produced a statistically significant response in both mutant and control mice. We have labeled the statistical significance in the Figures 4-7. Thank you for pointing this out.
Line 312: Optogenetic stimulation induced an increase from 130 to 180 breaths per min (Abbott et al., EJN 2014). It is surprising that this is called "modest". Baseline respiratory frequency was presented.
Thank you for the point. The word “modest” has been removed and the discussion has been changed accordingly (line 355-360).
Line 338: This discussion is not sufficiently nuanced. It is the increased Dia amplitude (to KCN only, not 10%CO2 ) and the stimulation of active expiration, to both stimuli, that is blocked by kyn in pFRG. There is no effect of breathing frequency. The current study would not detect such differences in active expiration.
Thank you for the suggestion. The discussion has been modified accordingly (line 382-388).
