Role of the postinspiratory complex in regulating swallow–breathing coordination and other laryngeal behaviors
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The findings in this study are important, as this brainstem region is implicated in a multitude of functions. The experimental procedures are difficult to implement and the preparation used and the skill required are impressive. The methods and data are solid, however, some analyses are incomplete, and the strength of evidence is also incomplete because the claims are only partially supported by the data. This work will interest those who study respiration, airway protection, and other oral behaviors.
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Abstract
Breathing needs to be tightly coordinated with upper airway behaviors, such as swallowing. Discoordination leads to aspiration pneumonia, the leading cause of death in neurodegenerative disease. Here, we study the role of the postinspiratory complex (PiCo) in coordinating breathing and swallowing. Using optogenetic approaches in freely breathing anesthetized ChATcre:Ai32, Vglut2cre:Ai32 and intersectional recombination of ChATcre:Vglut2FlpO:ChR2 mice reveals PiCo mediates airway protective behaviors. Activation of PiCo during inspiration or the beginning of postinspiration triggers swallow behavior in an all-or-nothing manner, while there is a higher probability for stimulating only laryngeal activation when activated further into expiration. Laryngeal activation is dependent on stimulation duration. Sufficient bilateral PiCo activation is necessary for preserving the physiological swallow motor sequence since activation of only a few PiCo neurons or unilateral activation leads to blurred upper airway behavioral responses. We believe PiCo acts as an interface between the swallow pattern generator and the preBötzinger complex to coordinate swallow and breathing. Investigating PiCo’s role in swallow and laryngeal coordination will aid in understanding discoordination with breathing in neurological diseases.
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Author Response
Reviewer #1 (Public Review):
This study used intersectional genetic approaches to stimulate a specific brainstem region while recording swallow/laryngeal motor responses. These results, coupled with histology, demonstrate that the PiCo region of the IRt mediates swallow/laryngeal behaviors, and their coordination with breathing. The data were gathered using solid methods and difficult electrophysiological techniques. This study and its findings are interesting and relevant. The analysis (and/or the presentation of the analysis) is incomplete, as there are analyses that need to be added to the manuscript. The interpretation of the data is mostly valid, but there are claims that are too speculative and are not well-supported by the results. The introduction and discussion would benefit from more citations and a deeper …
Author Response
Reviewer #1 (Public Review):
This study used intersectional genetic approaches to stimulate a specific brainstem region while recording swallow/laryngeal motor responses. These results, coupled with histology, demonstrate that the PiCo region of the IRt mediates swallow/laryngeal behaviors, and their coordination with breathing. The data were gathered using solid methods and difficult electrophysiological techniques. This study and its findings are interesting and relevant. The analysis (and/or the presentation of the analysis) is incomplete, as there are analyses that need to be added to the manuscript. The interpretation of the data is mostly valid, but there are claims that are too speculative and are not well-supported by the results. The introduction and discussion would benefit from more citations and a deeper exploration of how this study relates to other work - especially a thorough accounting of and comparison to other studies concerning putative swallow gates.
General/major concerns:
The field of respiratory control is far from unified regarding the role of PiCo in breathing or any other laryngeal behaviors. If anything, the current consensus does not support the triple-oscillator hypothesis (in which PiCo is one of 3 essential respiratory oscillators). The name "PiCo", short for "post-inspiratory complex", suggests a function that has not been well-supported by data - it is a putative post-inspiratory complex, at best. I suggest putting this area in context with other discussions i.e. IRt (such as in Toor et al., 2019) or Dhingra et al. 2020 showed broad activation of many brainstem sites at the post-I period (including pons, BotC, NTS)
The reviewer’s comment refers to our previous publication and not the present one. With all due respect to the reviewer, the submitted study investigates PiCo’s involvement in swallow and laryngeal activation and its coordination with breathing.
We did not feel that it is appropriate for us to critique the Dhingra paper in the present study. However, since this seems to be important to this reviewer, we would like to clarify: Because of filter characteristics, and the low temporal and spatial resolution of these field recordings, the approach used by Dhingra is inappropriate for providing insights into the presence or absence of PiCo. We therefore developed an alternative approach, which provides more detailed insights into population activity, the Neuropixel approach. This Neuropixel recording from PiCo (black trace) exemplifies how field recordings (yellow) fail to pick up post-I activity. We could provide many more examples, but as stated above, addressing the study by Dhingra is tangential to the present study.
We would also emphasize that the study by Dhingra was never designed to provide negative evidence, and Dhingra et al. never claimed that their study demonstrates the absence of PiCo. Unfortunately, the data by Dhingra were misinterpreted by Swen Hülsmann in his Journal of Physiology editorial which created considerable confusion, but also sensation in the field. Objectively, Toor et al reproduced the Anderson study in rats as we will elaborate below. Unfortunately, Toor et al added to the confusion, by renaming the PiCo area into IRt. The field of respiration would have also been confused if the first study reproducing the Smith et al. 1991 study in a different rodent species would have refused to call this area preBötC and instead would have called it e.g. ventrolateral reticular field.
Did you perform control experiments in which the opto stimulations were done on animals without the genetic channels (for example, WT or uncrossed ChAT-ires-cre, etc.), or in mice with the genetic channels that weren't crossed (uncrossed Ai32 mice)? If so, please include. If not, why?
Yes, we performed many control experiments. Aside of many recordings in which viral injections were targeted outside PiCo, we also performed optogenetic stimulations in mice lacking channelrhodopsin. We have now added the following statements and supplemental figure.
Optogenetic stimulation in mice lacking channelrhodopsin
Stimulation of PiCo, across all stimulation durations, in 3 Ai32+/+ mice and 4 ChATcre:Vglut2FlpO:ChR2 mice where the ChR2 did not transfect ChATcre:Vglut2FlpO, as confirmed by a post-hoc histological analysis, resulted in no response (Fig. S3).
How do you know that your opto activations simulate physiological activation? First, the intensive optical activation at the stim site does not occur in those neurons naturally.
This seems like a generic critique of the optogenetic approach. In none of the 10,000+ published optogenetic studies is it known to what extent optogenetic activation stimulates exactly the same neurons and the same degree of activity as during a natural behavior. What we know is that PiCo neurons are activated during postinspiration (Anderson et al. 2016) and that optogenetic activation stimulates these neurons and that this activation evokes the same muscles in the same temporal sequence as a water-evoked swallow. We assume that the reviewer’s comment does not intend to imply that “swallows” evoked by nonspecifically stimulating the SLN is more physiological than the optogenetically-evoked swallows of a specific neuron population? From the reviewer’s other comments, it is obvious that the reviewer has no problems with the results of the Toor study that used exclusively SLN stimulations, an approach which is known to be very non-specific.
Doing a natural (water) stim for comparison is good, but it cannot necessarily be directly compared to the opto stim. The water stim would activate many other brainstem regions in addition to PiCo.
Can the reviewer provide any hard evidence that “many other brainstem regions” are activated by water stimulation in comparison to optogenetic stimulation?
A caveat is that opto PiCo stim =/= water stim (in terms of underlying mechanisms) should be included. Second, in looking at the differences between water vs opto swallows in Table S2: it appears that the ChAT animals (S2A) have something weaker than a swallow with opto stim. For the Vglut2 and ChAT/Vglut2 (S2B&C), the opto swallows also aren't as "strong" as the water swallows (the X and EMG amplitudes are smaller). The interpretation/discussion attributes this to the lack of sensory input during opto stim, but does not mention the strong possibility that there is a difference in central mechanisms occurring. It also seems to be dismissed with the characterization of the swallow as "all-or-none" (see note on Fig 3 results).
With all due respect, we are somewhat surprised that the reviewer dismisses the entire paragraph in the discussion that specifically addresses the comparison between water-swallows and PiCo-stimulated swallows. We discussed the possibility that PiCo stimulated swallows may not activate the full pathway/mechanism as does the water swallow. We carefully compared and confirmed that PiCo-stimulated swallows have the same temporal motor sequence of the same muscles as those activated in water swallows. As already stated, it is surprising that the reviewer has no problem with accepting the validity of previously published methods like electrical non-specific stimulations of the cNTS or SLN, a frequently used and accepted model to produce and study swallow.
The writing needs extensive copy editing to improve clarity and precision, and to fix errors.
Thank you for this comment, we have revised and reviewed the writing.
Results/Fig 1: What proportion had no/other motor response (non-swallow, non-laryngeal) to the opto stim? I can extrapolate by subtraction, but it would be nice to see the "no/other response" on the plot.
With all due respect to this reviewer, but it is not possible to address this question. Specifically, it is not possible to know if a “No response” (meaning “no behavioral output” occurred in response to PiCo stimulation), would have resulted in a swallow or laryngeal activation. However, figure 2 contains responses other than swallows, i.e. “non swallows”, which includes both laryngeal activation as well as “no responses” meaning “no behavioral response” in response to PiCo stimulation. This was determined to assess how the respiratory rhythm is affected when a swallow is not produced by PiCo stimulation.
The explanation of genetics is too spread out and confusing. There needs to be more detail about all the genetic tools used, using the standard language for such tools, in one spot. Please also provide a clear explanation of what those tools accomplish. Include a figure if necessary.
We apologize for creating confusion. We added more explanations to the text.
Pick a conventional designator/abbreviation for the different strains, define them in the methods and in the first paragraph of the results section, and use those abbreviations throughout. I think that using ChAT as an abbreviation for your ChAT-ires-cre x Ai32 mice is confusing because it makes it sound like you're talking about the enzyme rather than the specific strain/neurons. Saying "ChAT stimulated swallows... swallows evoked by water or ChAT" makes it sound like the enzyme choline acetyltransferase itself is stimulating swallow. As is convention, pick a more precise abbreviation like ChAT-cre/Ai32 or ChAT:Ai32 or ChAT-ChR2 or ChAT/EYFP. This goes for the other strains as well.
Thank you for pointing this out. To avoid confusion the strains/neurons are now referred to as: ChATcre:Ai32, Vglut2cre:Ai32, and ChATcre:Vglut2FlpO:ChR2
For Fig S2C&D, why does it say mCherry? Isn't it tdTomato? Is it just an anti-ChAT antibody and then the tdTomato Ai65 is only labeling Vglut2? I don't see this in the methods section.
Thank you for pointing this out. We apologize for our mistake, and we have corrected the manuscript to say tdTomato.
I also don't see methods for all the staining in Fig S3. The photomicrograph says Vglut2-cre Ai6, but there's no mention of Ai6 anywhere else. Which mice are these? Did you cross Vglut2-cre with an Ai6 reporter mouse? How can you image an Ai6 mouse (which I assume expresses ZsGreen? and that you excited at 488?) and a 488 anti-goat in the same section (that's the only secondary listed in the methods that would work with your goat anti-ChAT)? Is there an error in listing the fluorophores in the methods? Please give more details on the microscopy including which filters were used for the triple staining.
We have decided to remove the CTb data from the manuscript.
Regarding the staining: I would expect the staining/maps in for the 2 different ChAT/Vglut2 intersectional strains to be similar (Fig 5A/B and S2C/D). The photomicrographs look very different to me, while the heat maps (this goes for all the heat maps in the paper) have barely distinguishable differences. In Fig 5, the staining looks much stronger than in Fig S2C. Why does it look like there are so many more transfected neurons in Fig 5A2 than there are red neurons in the corresponding panel Fig S2C2? And for Fig 5A4 and Fig S2C44? The plot and results text for Fig 5 says the avg number of neurons was 123+¬11. The plot for Fig S2D says 112+¬15, but the results text says 242+¬12 (not sure which is the correct number).
Thank you for your comments. Previously the heat maps had different scale bars if you compare Fig 5A/B and S2C/D (now figure S4C/D). We changed the heat maps keeping the same scale for all of them. Discussing the representative photomicrography, even figure Fig 5A/B and S4C/D represents the same cluster of cells (PiCo Chat/Vglut+). Figure S4D states 242 ± 12 neurons (also stated in the results section).
However, we want to point out that there are several technical differences between both, 1) figure 5A represents the transfection promoted by the virus injection, impacting the number of cells stained/transfected (133 ± 16 neurons), 2) figure S4C/D represents a intersectional mouse ChATcre: Vglut2FlpO: Ai65; (242 ± 12 neurons). In this case, we have more tdTomato positive cells because this genetic approach is able to detect most of the Chat and Vglut2 cells. The difference between figures is considered normal for anatomical studies, in some studies the same bregma can show different number of cells. Thus, the differences are due to the differences in the type of approaches (viral expressions vs. intersectional approach).
We have also added additional experiments to figure 5 (now N=7) which has been reflected in the text and figures.
The results text for Fig S2C also says the staining is "similar to the previous ChAT staining...", which I assume refers to S2A/B. The plot and results text for Fig S2B reports 403+¬39 neurons, while S2D is either 112 or 242 (not sure?). The plots have different Y scales, which should be changed to be the same. But why do the photomicrographs and the heat maps look so similar? I would expect far fewer neurons to be stained in the intersectional mice (Fig 5 and Fig S2C/D) than in the ChAT staining (Fig S2A/B). I am having trouble reconciling the different presentations/quantifications and making sense of the data in these histology figures.
We removed “similar to the previous ChAT staining” and we have reviewed the heat maps. Since the original submission, we performed more experiments and now added more animals to the analysis (now N=7), each heat map represents the correct number of neurons in PiCo, respectively to each experiment.
The Y scales has been adjust to better demonstrate the Chat staining vs. the intersectional mice triple conditioned.
How can you distinguish PiCo from non-PiCo in the histology, especially in the ChAT-only staining? It seems that you have arbitrarily defined the PiCo region, and only counted neurons within that very constrained area.
Even in ChAT-only staining, the N.ambiguus is very distinct from the cholinergic neurons located more medial to the N.ambiguus. This can be unambiguously be confirmed by combining ChAT with glutamatergic in situ staining as done in the Anderson et al. study, or unambiguously be demonstrated with the viral approach as done in the present study. Thus, we don’t see why it is arbitrary to define the distribution of PiCo neurons. What is arbitrary is the definition of the preBötC, yet the field of respiration seems to have no problem with this. We assume that the reviewer knows that Dbx1 neurons are spread along the entire ventral respiratory column and dorsal portion of the PreBötzinger Complex up to the level of the XII nucleus. Yet it is commonly accepted for authors to refer to the PreBötzinger Complex by counting dbx1 neurons within a constrained area of what is believed to be the PreBötzinger Complex, even though the borders are arbitrary. It is e.g. known that some of the ventrally located preBötC neurons are presumed rhythmogenic while the more dorsally located Dbx1 neurons are premotor. The transition from rhythmogenic to premotor is gradual. Similarly, NK1 staining, or SST staining is not restricted to the preBötC and it is arbitrary to define where preBötC begins and what to include. Indeed, our PNAS paper indicates that inspiratory bursts can be generated by optogenetically stimulating Dbx1 neurons along the entire VRC column – so it is not clear where the rhythmogenic portion of the preBötC begins rostrocaudally and dorsoventrally and where the rhythmogenic portion and preBötC itself ends. Thus, we want to re-iterate and emphasize, that for the present study, we developed a method using the cre/FlpO approach to unambiguously define the PiCo region. It is surprising that this reviewer does not acknowledge this technical advance that added significantly more specificity to the anatomical and physiological characterization of PiCo, than the Toor et al. study, and also the Anderson et al. study.
I can see stained neurons in the area immediately outside of PiCo, and I'd like to see lower-magnification images that show the staining distribution in a broader region surrounding PiCo as well, especially in the rest of the reticular formation.
We characterized the PiCo area based on the histological phenotype and in vitro and in vivo experiments performed by Anderson et al., 2016. PiCo is an area located close to the NAmb, presenting the same ChATcre phenotype. As stated above, the distribution and agglomeration of the NAmb is clearly very compact, and different then the observed ChATcre: Vglut2FlpO: Ai65 neurons located outside of NAmb. It is also important to emphasize, that like is the case for the preBötC, other transmitter phenotypes of neurons are also present in the PiCo region (i.e. GABA or Dbx1). However, the study performed by Anderson et al, 2016 paper, described only the functions of cholinergic neurons located in PiCo, and we always planned to publish a paper of the other neurons within PiCo – this area e.g. contains pacemaker neurons etc. But, I hope that the reviewer acknowledges that many investigators have studied the preBötC for the past 30 years. Hence, much more information has been accumulated on this region (which btw was at least as controversial at the beginning), and it will likely take at least another 30 years to fully identify and characterize PiCo.
Similarly, how can you be sure you're stereotaxically targeting PiCo precisely (600um in diameter?) with your opto fiber (200um in diameter). Wouldn't small variations in anatomy put the fiber outside the tiny PiCo area?
We assume the reviewer means “stereotactically”. And yes, the reviewer is correct, it is necessary to position the laser at a consistent anatomical location. Placement of the optical fibers outside of this area does not result in activation of PiCo. We have added an additional supplemental figure (Figure S6) to address this.
Please put N's and stats results in Table S1 for both swallow and laryngeal activity. I took what I assume to be the Ns (10, 11, and 4) and did some stats like the ones you presented for the laryngeal duration. The differences between vagus duration for 40 and 200 ms pulse durations are all significant for each strain, by my calculations. Also, I think there must be an error in the orange swallow plot in Fig 3A. The orange dots don't correspond to the table values. I plotted all the Table S1 values for each strain. Each line looks similar to the blue laryngeal activation plot in Fig 3A. The slopes of the Vglut2 were less than the other strains, and the slopes for the swallow behavior were less than the laryngeal behavior for all strains. Otherwise, they all look similar. Please double-check your values/stats to address these discrepancies. If it is indeed true that the stim pulse duration affects swallow duration, revise the interpretations and manuscript accordingly.
We thank the reviewer for the diligence in reviewing our manuscript. But, with all due respect, the reviewer is incorrect and misunderstood the data. To clarify: Table S1 is only presenting data for laryngeal activation, swallow data is presented in Table S2. The orange data points in Fig 3A are not detailed in Table S1 or S2. Table S2 is the average of all swallows across all laser pulse durations since the laser pulse duration does not affect swallow behavior duration. All data will be publically available after publication of the manuscript.
Figure 3A is only representing the ChATcre:Vglut2FlpO:ChR2 column of Table S1
The N’s have been added to table S1
Please add more details on stats in general, including the specific tests that were performed, F values and degrees of freedom, etc.
Thank you, this has been added throughout the results section. Please refer to the results section for this addition. However below we have provided an example.
An example: A two-way ANOVA revealed a significant interaction between time and behavior (p<0.0001, df= 4, F= 23.31) in ChATcre:Vglut2FlpO:ChR2 mice (N=7).
How do you know that you're not just activating motoneurons in the NA when you stimulate your ChAT animals, especially given the results in Fig 1B? In this case, the phase-specific results could be explained by inhibitory inputs (during inspiration) to motoneurons in the region of the opto stim.
As stated in this paper as well as the Anderson et al 2016 paper (and for that matter also the Toor et al study) this is a caveat. This major caveat motivated the development and use of the ChATcre:Vglut2FlpO:ChR2 (specifically targeting the PiCo neurons that co-express ChAT and Vglut2, not laryngeal motor neurons) experiments that have mostly the same response as the ChATcre:Ai32 mice. We cannot say this is due to inhibitory inputs to laryngeal motoneurons, since the cre/FlpO specific experiments are not directly activating laryngeal motoneurons. But we do not want to entirely exclude that some premotor mechanisms may also occur in PiCo. The reviewer may know that there is overlap of rhythmogenic and premotor functions for the Dbx1 neurons in the PreBötC, But, addressing this issue is beyond the scope of this study. In fact, we are working on a separate connectivity study using novel, still unpublished antegrade and retrograde vectors that do not reveal any direct connections to laryngeal motoneurons. Hence, we expect that the connectivity from PiCo to laryngeal motoneurons is more complex and addressing this question cannot be done as a simple add-on to an already complex study. Again, we would refer to the PreBötzinger complex, where nobody expects that one study can resolve all the physiological and anatomical characterizations that have been accumulated over 30 years in one study. We would argue that in some ways, our cre/FlpO approach is more specific than the Dbx1 stimulations which activates not only rhythmogenetic PreBötzinger complex neurons, but also pre motoneurons as well as glia cells, and many neurons rostral and caudal to the PreBötzinger complex. We are aware of these caveats, and we have discussed this in the original submission, and also in the revision.
While the study from Toor et al is cited, there needs to be a much more thorough discussion of how their findings relate to the current study.
Many thanks for asking for a more thorough discussion of Toor et al., which we are happy to provide here. Perhaps we were too polite in our original manuscript to emphasize all the problems in that study.
They demonstrated that PiCo isn't necessary for the apneic portion of swallow. Inhibiting this region also didn't affect TI.
Please note – the fact that Toor et al did not find an effect on TI confirms Anderson et al. 2016: In Figure 3G,3F of the Nature paper, the reviewer will find that injections of DAMGO and SST into PiCo inhibited post-I activity without affect inspiratory duration. This figure also shows that the inspiratory burst can terminate in the absence of postinspiratory activity.
The reviewer states: “They demonstrated that PiCo isn't necessary for the apneic portion of swallow”. With all due respect to this reviewer, this is NOT correct. Toor et al showed that inhibiting PiCo did block SLN-evoked fictive-swallows but not the apnea caused by SLN stimulation. This is not the apnea caused by swallows (which was never studied by Toor), but by the SLN stimulation. The apnea evoked by SLN stimulation has most likely nothing to do with the apnea caused by swallows. Unfortunately, the Toor et al. makes the same misleading claim as the reviewer.
PiCo cannot be the sole source of post-I timing, and the evidence overwhelmingly favors the major involvement of other regions such as the pons.
This comment seems to be unrelated to the main thrust of this paper that studies PiCo’s involvement in swallow and laryngeal activation in coordination with breathing. However, since this comment seems to discredit the Ramirez lab in general, we would like to clarify that inhibiting PiCo with DAMGO and SST inhibits post-I activity (Anderson et al 2016, Fig.3G,3F). Thus, we don’t understand the rationale or actual data for the reviewer’s conclusion that PiCo cannot be the sole source of post-I timing? We also don’t understand the basis for the reviewer’s conclusion that “the evidence overwhelmingly favors the major involvement of other regions such as the pons”. We also want to add, that no-where in the Anderson et al. study did we state that the pons plays NO role. Indeed, we specifically stated: “In this context it will be interesting to resolve the role of the PiCo in specific postinspiratory behaviors and to identify how the PiCo interacts with other neural networks such as the Kolliker-Fuse nucleus, a pontine structure that has been hypothesized to gate postinspiratory activity and the periaqueductal grey a structure involved in vocalization and the control of postinspiration”.
They also showed that inhibition of all neurons (not just ChAT/Vglut) in the PiCo region suppresses post-I activity in eupnea. This suppression was overcome by the increased respiratory drive during hypoxia.
Before comparisons are made with Toor et al. it is important to note the species and methodological differences between Toor et al. rat anesthetized, vagotomized, paralyzed and artificially ventilated model which evaluated fictive swallows (deafferented and paralyzed). By contrast this study uses a mouse anesthetized, vagal intact, freely breathing model and evaluates natural physiologic swallow via water and central stimulation. It seems that the reviewers does not acknowledge one of the main innovations of this study. For this study we introduced a genetic approach to specifically target and activate ChATcre/Vglut2FlpO PiCo neurons. This has never been done before, and developing this approach took more than 4 years of breeding and crossing and testing different options.
As for Toor et al., these authors pharmacologically, bilaterally inhibited neurons in the area of PiCo with isoguvacine, a specific GABA-A agonist. Even though this pharmacological intervention does not specifically inhibit cholinergic/glutamatergic neurons in PiCo, these authors essentially confirm the study by Anderson et al. We do not find this finding controversial. Perhaps the reviewer finds the definition of PiCo “controversial”, because Toor et al called the identical area IRt instead of PiCo, even though they exactly reproduce the finding by Anderson. Toor et al. not only arrive at the same conclusion as Anderson but they added more details – none of which is contradicting the results by Anderson et al.: Here are excerpts from the Toor study “We therefore conclude that the ongoing activity of neurons in the IRt contributes to eupneic respiratory and sympathetic post-I activities without exerting significant control on other respiratory or cardiovascular parameters” “IRt significantly inhibited the post-I components of VNA” “IRt inhibition was also associated with a reduction in PNA” “increase in respiratory cycle frequency” “due to a reduction in TE“ “with no effect on TI observed”. “Bilateral microinjection of isoguvacine selectively reduced the magnitudes of post-I VNA and rSNA, but not PNA responses to acute hypoxemia”.
In this statement the reviewer probably refers to one particular aspect, i.e. the fact that Toor et al. did not significantly block some of the post-I activity – they state: “had no significant effect on the AUC of post-I rSNA (305+/- 24 vs 230+/- 28,p=0.16,n=6)”. Please note that there is a tendency, a reduction from 305-230. Perhaps the Toor study was not sufficiently powered to fully block the effect, perhaps the drug did not inhibit the entire PiCo. These are all open questions that a critical reader should know. The reviewer will agree that it is as difficult if not more difficult to demonstrate the absence of an effect. To arrive at a negative conclusion experiments should be done with the same scrutiny than to demonstrate a positive result. We also assume that the reviewer is familiar with animal experiments and will understand that pharmacological injections are often difficult to interpret, in particular in case of local in vivo injections. It is possible that Toor et al is inhibiting e.g. parts of the Bötzinger complex.
We have added to the manuscript the following statement: It is important to note that SLN stimulation does not only trigger swallows, but also changes in the overall stiffness and tension of the vocal cords (Chhetri et al., 2013) as well as prolonged hypoglossal activation independent of swallowing (Jiang, Mitchell, & Lipski, 1991). It has been hypothesized that inhibition of the IRt blocks fictive swallow but not swallow-related apnea. Yet this apnea was generated by SLN stimulation and not by a natural swallow stimulation (Ain Summan Toor et al., 2019). It is known that SLN stimulation causes endogenous release of adenosine that activates 2A receptors on GABAergic neurons resulting in the release of GABA on inspiratory neurons and subsequent inspiratory inhibition (Abu-Shaweesh, 2007), suggesting that the SLN evoked apnea may not be the same as a swallow related apnea. Moreover, microinjections of isoguvacine into the Bötzinger complex attenuated the apneic response but not the ELM burst activity (Sun, Bautista, Berkowitz, Zhao, & Pilowsky, 2011), suggesting the Bötzinger complex, not PiCo, could be involved in modulating apnea.
We would also like to add that our current study characterized swallow-related specific muscles and nerves in both water-triggered and PiCo-triggered swallows to better characterize the physiological properties of this swallow behavior. By contrast, Toor et al. only characterized nerve activities that are involved in multiple upper airway activities and breathing. It is somewhat surprising that the reviewer did not consider the fact that Toor et al. characterized putative swallows that were triggered by SLN stimulation and that Toor et al. were content with nerve-recordings and failed to confirm that the behavior that they evoked is actually a physiological swallow. Which, according to the comments from this reviewer (see above), indicates the possibility of differences in central mechanisms occurring between fictive swallow and physiological swallows.
While we have cited Toor et al and their truly excellent work in the broad iRt we did not feel it is appropriate to critique them for the fact that they are confusing the field by using a different anatomical term for the area that was clearly defined by us as an area containing cholinergic-glutamatergic neurons. We also did not feel it is appropriate to discuss results that are similar to comparing Apples and Oranges. Toor et al. never specifically manipulated glutamatergic-cholinergic neurons, thus their entire results rest on indirect stimulation affecting this general area – which will unavoidably also include laryngeal motoneurons. We don’t want to criticize this approach, since PiCo is heterogenous, which is another misunderstanding that we find in the reviewers’ critique. We used cholinergic-glutamatergic neurons to define this area. However, like the preBötC, PiCo is also heterogenous. This region contains inhibitory neurons, it also contains glutamatergic neurons that are not cholinergic, and cholinergic neurons that are not glutamatergic. Because of this heterogeneity we compared the effects of stimulating glutamatergic neurons and cholinergic neurons as well as cholinergic-glutamatergic neurons. This is an approach that is generally accepted in the field. As already stated, there is not a single marker that uniquely characterizes the PreBötC. Thus, when stimulating Dbx1 neurons, glutamatergic neurons, or Somatostatin neurons it only captures subpopulations of this region. The recently published study by Menuet et al. in eLife, used even more indirect methods to inhibit preBötC. They used a pan-neuronal CBA promotor that targets neurons irrespective of phenotype. It is not our intention to discredit this very elegant study, but we object the statement that we “have arbitrarily defined the PiCo region”.
This study has not demonstrated some of the things that are depicted in Fig 7 and included in the discussion. While swallow can inhibit inspiration, there are many mechanisms by which this can happen other than a direct inhibitory connection from the DGS to PreBotC. You cite Sun et al., 2011 findings of "a group of neurons that inhibits inspiration" during SLN stim, but don't mention that it is the BotC and that the paper shows that swallow apnea is dependent on BotC. That is also supported by the Toor study. I don't understand how post-I (aka E) can be discussed without discussion of the BotC - this is a glaring omission.
We have removed figure 7, which was only meant as a hypothetical schematic.
Why is it necessary for PiCo to innervate the cNTS?
This was a hypothesis based on CTb data that we have now removed.
That is true if the conjecture that PiCo gates swallowing is true, as the cNTS is the only known region for central swallow gating. However, PiCo could influence afferent input to the NTS less directly, and therefore not function as a gating hub per se. The experimental evidence does not warrant the claim that PiCo gates swallowing. The definition of a swallow gate(s) is a topic of much debate and no conclusive experimental evidence has emerged for swallow gating regions to exist anywhere except in the NTS. The current study's evidence also does not meet the criteria necessary to conclusively call PiCo a swallow gate. The authors should soften this claim and language throughout the manuscript.
Although we do not know of any studies that has optogenetically gated swallow in the cNTS, it seems the reviewer objects our use of the word “gate”. We have revised the manuscript and removed any wording stating PiCo is a swallow “gate”. It would be interesting to know whether the reviewer has the same objections of the use of the word “relay” as done by Toor et al.?
It is also unclear that PiCo acts directly on the swallow pattern generator to gate swallowing. It is not just "conceivable that the gating mechanism involves" the pons, but nearly certain. Swallow gating by respiratory activity may not be able to be ascribed to one particular location. At a minimum, it likely involves the NTS/DSG, pons, and possibly IRt (inclusive of PiCo). The authors are correct that "further studies are necessary to understand the interaction between PiCo and the pontine respiratory group on the gating swallow and other airway protective behaviors." This is why it shouldn't be stated that "this small brainstem microcircuits acts as a central gating mechanism for airway protective behaviors."
We have removed all language stating PiCo is a swallow gate.
PiCo is likely part of the VSG (and thus the swallow pattern generator). PiCo, as part of the IRt/VSG could indeed be surveilling afferent information and providing output that affects swallow or other laryngeal activation and the coordination of these behaviors with breathing. However, this is not the responsibility of PiCo alone. This role is likely shared by other parts of the SPG, and may require distributed SPG network participation to be functional. If one were to stim other regions of the distributed SPG, similar results might be expected. When Toor et al silenced the PiCo area (and locations that lie at least lightly beyond the borders of what the present study defines as PiCo), stim-evoked fictive swallows were greatly suppressed. However, swallow-related apnea was unaffected. This supports the role of PiCo as a premotor relay for swallow motor activation, but not as the site that terminates inspiration. Therefore, it cannot be called a gate.
We already addressed the issue that Toor never demonstrated that the “swallow-related” apnea was unaffected. Toor et al only demonstrated that the SLN-evoked apneas were unaffected, and their conclusions were only based on nerve recordings under fictive conditions (deafferented and paralyzed). Also, to the best of our knowledge, many aspects of the putative swallow pattern generator that this reviewer mentions are purely hypothetical. However, to avoid further arguments, we have removed the word gate and Figure 7 from this manuscript.
Similarly, Fig 7 does not accurately depict things that are already well-supported by evidence. PiCo should be included as part of the swallow pattern generator (VSG), not as a separate entity acting on it. The BotC and pons are glaring omissions. This study has not demonstrated the labeled inhibitory connection from DSG to PreBotC. The legend states speculations as fact and needs to be dialed way back to either include statements with solid experimental evidence or to clearly mark things as putative/speculative.
We have removed figure 7.
The discussion of expiratory laryngeal motoneurons needs to be expanded and integrated better into the discussion of swallow, post-I, and other laryngeal motor activation. Why can't PiCo just be premotor to ELMs?
If PiCo would “only” or “just” be premotor to ELM then it would not be expected that it could trigger an all-or-none swallow response with a temporal activity pattern similar to the one of a water-evoked swallow. We would also not expect that the activation of the activity pattern is independent of the laser stimulation duration as demonstrated in Figure 3. This was our reasoning why we originally called PiCo a “gate” because at the correct phase it will gate/trigger a complex swallow sequence. But, as stated above, we avoid the word gate in the revised manuscript.
Concerning the discussion of "PiCo's influence as a gate for airway protective behaviors is blurred...": The incomplete swallow motor sequence didn't seem super different in timing or duration compared to the fully transfected animals (comparing plots from Fig 6 to Fig S1, and Table S2 to Table S3. The values for swallow durations (XII and X) for each group for water and opto seem within similar ranges, as do the differences between water & opto-evoked swallows between strains. While the motor pattern is distinctive from the normal swallow, with laryngeal activity rather than submental activity leading, one might not even be able to call that a swallow. Is it evidence against a classic all-or-nothing swallow behavior any more than the graded swallow results from (fully transfected) Table S1?
We fully agree that it is possible that this unidentified behavior may not be a swallow. We have changed the name of this behavior to “upper airway motor activity.” However we also cannot rule out the possibility of this being some portion of a graded swallow which would argue that a graded swallow response is exact evidence against the classic all or nothing swallow behavior.
Please expand on this point and put it into context with others' results: "This brings into question whether this is the first evidence against the classic dogma of swallow as an "all or nothing" behavior, and/or whether this is an indication that activating the cholinergic/glutamatergic neurons in PiCo is not only gating the SPG, but is actually involved in assembling the swallow motor pattern itself."
This has been expanded and included citation of other studies. The following paragraph can be found in the discussion
Swallow has been thought of as an “all or nothing” response as early as 1883 (Meltzer, 1883). Whether modulating spinal or vagal feedback (Huff A, 2020b), central drive for swallow/breathing (Huff, Karlen-Amarante, Pitts, & Ramirez, 2022) or lesions in swallow related areas of the brainstem (Car, 1979; Robert W Doty, Richmond, & Storey, 1967; Wang & Bieger, 1991) swallow either occurred or did not. Swallows are thought to be a fixed action pattern, with duration of stimulation having no effect on behavior duration (Fig. 3) (Dick, Oku, Romaniuk, & Cherniack, 1993). Thus, it was particularly interesting that in instances when few PiCo neurons were transfected, either unilateral or bilateral, an unknown activation of upper airway activity occurred. Motor activity no longer outlasted laser stimulation rather was contained within, and the timing of the motor sequence was reversed in comparison to a water or PiCo evoked swallow (Fig. 6). Thus, if insufficient numbers of neurons are activated, PiCo’s influence to specifically activate swallow or laryngeal activation is blurred, resulting in the uncoordinated activation of muscles involved in both behaviors. This brings possible evidence against the classic dogma of swallow as an “all or nothing” behavior, or the presence of an entirely different behavior. We are not the first to bring possible evidence against the classic dogma, “small swallows” were described but failed to be discovered if this was in-fact a partial or incomplete swallow (Miller & Sherrington, 1915). The SPG is thought to consist of bilateral circuits (hemi-CPGs) that govern ipsilateral motor activities, but receive crossing inputs from contralateral swallow interneurons in the reticular formation, thought to coordinate synchrony of swallow movements (Kinoshita et al., 2021; Sugimoto, Umezaki, Takagi, Narikawa, & Shin, 1998; Sugiyama et al., 2011). Incomplete activation of PiCo activates the muscular components of a swallow, without establishing the coordinated timing and sequence of the pattern. It is possible that PiCo is involved in assembling the swallow motor pattern itself and unilateral activation of PiCo could either desynchronize swallow interneurons or activates only one side of the SPG. Since we did not record bilateral swallow related muscles and nerves this question needs to be further examined.
Reviewer #3 (Public Review):
Huff et.al further characterise the anatomy and function of a population of excitatory medullary neurons, the Post-inspiratory Complex (PiCo), which they first described in 2016 as the origin of the laryngeal adduction that occurs in the post-inspiratory phase of quiet breathing. They propose an additional role for the glutamatergic and cholinergic PiCo interneurons in coordinating swallowing and protective airway reflexes with breathing, a critical function of the central respiratory apparatus, the neural mechanics of which have remained enigmatic. Using single allelic and intersectional allelic recombinase transgenic approaches, Huff et al. selectively excited choline acetyltransferase (ChAT) and vesicular glutamate transporter-2 (VGluT2) expressing neurons in the intermediate reticular nucleus of anesthetised mice using an optogenetic approach, evoking a stereotyped swallowing motor pattern (indistinguishable from a water-induced swallow) during the early phase of the breathing cycle (within the first 10% of the cycle) or tonic laryngeal adduction (which tracked tetanically with stimulus length) during the later phase of the breathing cycle (after 70% of the cycle).
They further refine the anatomical demarcation of the PiCo using a combination of ChAT immunohistochemistry and an intersectional transgenic strategy by which fluorescent reporter expression (tdTomato) is regulated by a combinatorial flippase and cre recombinase-dependent cassette in triple allelic mice (Vglut2-ires2-FLPO; ChAT-ires-cre; Ai65).
Lastly, they demonstrate that the PiCo is anatomically positioned to influence the induction of swallowing through a series of neuroanatomical experiments in which the retrograde tracer Cholera Toxin B (CTB) was transported from the proposed location of the putative swallowing pattern generator within the caudal nucleus of the solitary tract (NTS) to glutamatergic ChAT neurons located within the PiCo. We would like to thank the reviewer for acknowledging the technical advances of the present study and for the positive statements in general.
Methods and Results
The experimental approach is appropriate and at the cutting edge for the field: advanced neuroscience techniques for neuronal stimulation (virally driven opsin expression within a genetically intersecting subset of neurons) applied within a sophisticated in vivo preparation in the anaesthetized mouse with electrophysiological recordings from functionally discrete respiratory and swallowing muscles. This approach permits selective stimulation of target cell types and simultaneous assessment of gain-of-function on multiple respiratory and swallowing outputs. This intersectional method ensures PiCo activation occurs in isolation from surrounding glutamatergic IRt interneurons, which serve a diverse range of homeostatic and locomotor functions, and immediately adjacent cholinergic laryngeal motor neurons within the nucleus ambiguous (seen by some as a limitation of the original study that first described the PiCo and its roll in post-I rhythm generation Anderson et al., 2016 Nature 536, 76-80). These experiments are technically demanding and have been expertly performed.
Again, we would like to thank the reviewer for these positive comments acknowledging the advances of the present study.
The supplemental tracing experiments are of a lower standard. CTB is a robust retrograde tracer with some inherent limitations, paramount of which is the inadvertent labelling of neurons whose axons pass through the site of tracer deposition, commonly leading to false positives. In the context of labelling promiscuity by CTB, the small number of PiCo neurons labelled from the NTS (maybe 5 or 6 at most in an optical plane that features 20 or more PiCo neurons) is a concern. Even assuming that only a small subset of PiCo neurons makes this connection with the presumed swallowing CPG within the cNTS, interpretation is not helped by the low contrast of the tracer labelling (relative to the background) and the poor quality of the image itself. The connection the authors are trying to demonstrate between PiCo and the cNTS could be solidified using anterograde tracing data the authors should already have at hand (i.e. EYFP labelling driven by the con-fon AAV vectors from PiCo neurons (shown in Fig5), which should robustly label any projections to the cNTS).
We fully agree with the reviewer that the CTB staining is of a lower standard and have removed this approach.
The retrograde labelling from laryngeal muscles seems unnecessary: the laryngeal motor pool is well established (within the nAmb and ventral medulla), and it would be unprecedented for a population of glutamatergic neurons to form direct connections with muscles (beyond the sensory pool).
The authors support their claim that PiCo neurons gate laryngeal activity with breathing through the demonstration that selective activation of glutamatergic and cholinergic PiCo neurons is sufficient to drive oral/pharyngeal/laryngeal motor responses under anaesthesia and that such responses are qualitatively shaped by the phase of the respiratory cycle within which stimulation occurs. Optical stimulation within the first 10% of the respiratory cycle was sufficient to evoke a complete, stereotyped swallow that reset the breathing cycle, while stimuli within the later 70% of the cycle, evoked discharge of the laryngeal muscles in a stimulus length-dependent manner. Induced swallows were qualitatively indistinguishable from naturalistic swallow induced by the introduction of water into the oral cavity. The authors note that a detailed interpretation of induced laryngeal activity is probably beyond the technical limits of their recordings, but they speculate that this activity may represent the laryngeal adductor reflex. This seems like a reasonable conclusion.
We thank the reviewer for this comment. Unfortunately, we felt compelled to remove the word “gating” based on the statements by reviewer 1.
The authors propose a model whereby the PiCo impinges upon the swallowing CPG (itself a poorly resolved structure) to explain their physiological data. The anatomical data presented in this study (retrograde transport of CTB from cNTS to PiCo) are insufficient to support this claim. As suggested above, complementary, high-quality, anterograde tracing data demonstrating connectivity between these structures as well as other brain regions would help to support this claim and broaden the impact of the study.
We fully agree with this reviewer. We have been working on a thorough anatomical characterization for more than 3 years using cutting edge anterograde and retrograde viruses in collaboration with vector experts at the University of Irvine. But these are partly novel, unpublished techniques that are in development, and require many careful controls and characterization. We feel that this is a separate study as it doesn’t relate to swallowing coordination and also includes partly different authors. We hope to submit this as a separate study later this year.
This study proposes that the PiCo in addition to serving as the site of generation of the post-I rhythm also gates swallowing and respiration. The scope of the study is small, and limited to the subfields of swallowing and respiratory neuroscience, however, this is an important basic biological question within these fields. The basic biological mechanisms that link these two behaviors, breathing and swallowing, are elusive and are critical in understanding how the brain achieves robust regulation of motor patterning of the aerodigestive tract, a mechanism that prevents aspiration of food and drink during ingestion. This study pushes the PiCo as a key candidate and supports this claim with solid functional data. A more comprehensive study demonstrating the necessity of the PiCo for swallow/breathing coordination through loss of function experiments (inhibitory optogenetics applied in the same transgenic context) along with robust connectivity data would solidify this claim.
Thanks again for the positive assessment of our study.
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eLife assessment
The findings in this study are important, as this brainstem region is implicated in a multitude of functions. The experimental procedures are difficult to implement and the preparation used and the skill required are impressive. The methods and data are solid, however, some analyses are incomplete, and the strength of evidence is also incomplete because the claims are only partially supported by the data. This work will interest those who study respiration, airway protection, and other oral behaviors.
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Reviewer #1 (Public Review):
This study used intersectional genetic approaches to stimulate a specific brainstem region while recording swallow/laryngeal motor responses. These results, coupled with histology, demonstrate that the PiCo region of the IRt mediates swallow/laryngeal behaviors, and their coordination with breathing. The data were gathered using solid methods and difficult electrophysiological techniques. This study and its findings are interesting and relevant. The analysis (and/or the presentation of the analysis) is incomplete, as there are analyses that need to be added to the manuscript. The interpretation of the data is mostly valid, but there are claims that are too speculative and are not well-supported by the results. The introduction and discussion would benefit from more citations and a deeper exploration of how …
Reviewer #1 (Public Review):
This study used intersectional genetic approaches to stimulate a specific brainstem region while recording swallow/laryngeal motor responses. These results, coupled with histology, demonstrate that the PiCo region of the IRt mediates swallow/laryngeal behaviors, and their coordination with breathing. The data were gathered using solid methods and difficult electrophysiological techniques. This study and its findings are interesting and relevant. The analysis (and/or the presentation of the analysis) is incomplete, as there are analyses that need to be added to the manuscript. The interpretation of the data is mostly valid, but there are claims that are too speculative and are not well-supported by the results. The introduction and discussion would benefit from more citations and a deeper exploration of how this study relates to other work - especially a thorough accounting of and comparison to other studies concerning putative swallow gates.
General/major concerns:
• The field of respiratory control is far from unified regarding the role of PiCo in breathing or any other laryngeal behaviors. If anything, the current consensus does not support the triple-oscillator hypothesis (in which PiCo is one of 3 essential respiratory oscillators). The name "PiCo", short for "post-inspiratory complex", suggests a function that has not been well-supported by data - it is a putative post-inspiratory complex, at best. I suggest putting this area in context with other discussions i.e. IRt (such as in Toor et al., 2019) or Dhingra et al. 2020 showed broad activation of many brainstem sites at the post-I period (including pons, BotC, NTS)
• Did you perform control experiments in which the opto stimulations were done on animals without the genetic channels (for example, WT or uncrossed ChAT-ires-cre, etc.), or in mice with the genetic channels that weren't crossed (uncrossed Ai32 mice)? If so, please include. If not, why?
• How do you know that your opto activations simulate physiological activation? First, the intensive optical activation at the stim site does not occur in those neurons naturally. Doing a natural (water) stim for comparison is good, but it cannot necessarily be directly compared to the opto stim. The water stim would activate many other brainstem regions in addition to PiCo. A caveat is that opto PiCo stim =/= water stim (in terms of underlying mechanisms) should be included. Second, in looking at the differences between water vs opto swallows in Table S2: it appears that the ChAT animals (S2A) have something weaker than a swallow with opto stim. For the Vglut2 and ChAT/Vglut2 (S2B&C), the opto swallows also aren't as "strong" as the water swallows (the X and EMG amplitudes are smaller). The interpretation/discussion attributes this to the lack of sensory input during opto stim, but does not mention the strong possibility that there is a difference in central mechanisms occurring. It also seems to be dismissed with the characterization of the swallow as "all-or-none" (see note on Fig 3 results).
• The writing needs extensive copy editing to improve clarity and precision, and to fix errors.
• Results/Fig 1: What proportion had no/other motor response (non-swallow, non-laryngeal) to the opto stim? I can extrapolate by subtraction, but it would be nice to see the "no/other response" on the plot.
• The explanation of genetics is too spread out and confusing. There needs to be more detail about all the genetic tools used, using the standard language for such tools, in one spot. Please also provide a clear explanation of what those tools accomplish. Include a figure if necessary. Pick a conventional designator/abbreviation for the different strains, define them in the methods and in the first paragraph of the results section, and use those abbreviations throughout. I think that using ChAT as an abbreviation for your ChAT-ires-cre x Ai32 mice is confusing because it makes it sound like you're talking about the enzyme rather than the specific strain/neurons. Saying "ChAT stimulated swallows... swallows evoked by water or ChAT" makes it sound like the enzyme choline acetyltransferase itself is stimulating swallow. As is convention, pick a more precise abbreviation like ChAT-cre/Ai32 or ChAT:Ai32 or ChAT-ChR2 or ChAT/EYFP. This goes for the other strains as well.
• For Fig S2C&D, why does it say mCherry? Isn't it tdTomato? Is it just an anti-ChAT antibody and then the tdTomato Ai65 is only labeling Vglut2? I don't see this in the methods section.
• I also don't see methods for all the staining in Fig S3. The photomicrograph says Vglut2-cre Ai6, but there's no mention of Ai6 anywhere else. Which mice are these? Did you cross Vglut2-cre with an Ai6 reporter mouse? How can you image an Ai6 mouse (which I assume expresses ZsGreen? and that you excited at 488?) and a 488 anti-goat in the same section (that's the only secondary listed in the methods that would work with your goat anti-ChAT)? Is there an error in listing the fluorophores in the methods? Please give more details on the microscopy including which filters were used for the triple staining.
• Regarding the staining: I would expect the staining/maps in for the 2 different ChAT/Vglut2 intersectional strains to be similar (Fig 5A/B and S2C/D). The photomicrographs look very different to me, while the heat maps (this goes for all the heat maps in the paper) have barely distinguishable differences. In Fig 5, the staining looks much stronger than in Fig S2C. Why does it look like there are so many more transfected neurons in Fig 5A2 than there are red neurons in the corresponding panel Fig S2C2? And for Fig 5A4 and Fig S2C44? The plot and results text for Fig 5 says the avg number of neurons was 123+¬11. The plot for Fig S2D says 112+¬15, but the results text says 242+¬12 (not sure which is the correct number).
• The results text for Fig S2C also says the staining is "similar to the previous ChAT staining...", which I assume refers to S2A/B. The plot and results text for Fig S2B reports 403+¬39 neurons, while S2D is either 112 or 242 (not sure?). The plots have different Y scales, which should be changed to be the same. But why do the photomicrographs and the heat maps look so similar? I would expect far fewer neurons to be stained in the intersectional mice (Fig 5 and Fig S2C/D) than in the ChAT staining (Fig S2A/B). I am having trouble reconciling the different presentations/quantifications and making sense of the data in these histology figures.
• How can you distinguish PiCo from non-PiCo in the histology, especially in the ChAT-only staining? It seems that you have arbitrarily defined the PiCo region, and only counted neurons within that very constrained area. I can see stained neurons in the area immediately outside of PiCo, and I'd like to see lower-magnification images that show the staining distribution in a broader region surrounding PiCo as well, especially in the rest of the reticular formation.
• Similarly, how can you be sure you're stereotaxically targeting PiCo precisely (600um in diameter?) with your opto fiber (200um in diameter). Wouldn't small variations in anatomy put the fiber outside the tiny PiCo area?
• Please put N's and stats results in Table S1 for both swallow and laryngeal activity. I took what I assume to be the Ns (10, 11, and 4) and did some stats like the ones you presented for the laryngeal duration. The differences between vagus duration for 40 and 200 ms pulse durations are all significant for each strain, by my calculations. Also, I think there must be an error in the orange swallow plot in Fig 3A. The orange dots don't correspond to the table values. I plotted all the Table S1 values for each strain. Each line looks similar to the blue laryngeal activation plot in Fig 3A. The slopes of the Vglut2 were less than the other strains, and the slopes for the swallow behavior were less than the laryngeal behavior for all strains. Otherwise, they all look similar. Please double-check your values/stats to address these discrepancies. If it is indeed true that the stim pulse duration affects swallow duration, revise the interpretations and manuscript accordingly.
• Please add more details on stats in general, including the specific tests that were performed, F values and degrees of freedom, etc.
• How do you know that you're not just activating motoneurons in the NA when you stimulate your ChAT animals, especially given the results in Fig 1B? In this case, the phase-specific results could be explained by inhibitory inputs (during inspiration) to motoneurons in the region of the opto stim.
• While the study from Toor et al is cited, there needs to be a much more thorough discussion of how their findings relate to the current study. They demonstrated that PiCo isn't necessary for the apneic portion of swallow. Inhibiting this region also didn't affect TI. PiCo cannot be the sole source of post-I timing, and the evidence overwhelmingly favors the major involvement of other regions such as the pons. They also showed that inhibition of all neurons (not just ChAT/Vglut) in the PiCo region suppresses post-I activity in eupnea. This suppression was overcome by the increased respiratory drive during hypoxia.
• This study has not demonstrated some of the things that are depicted in Fig 7 and included in the discussion. While swallow can inhibit inspiration, there are many mechanisms by which this can happen other than a direct inhibitory connection from the DGS to PreBotC. You cite Sun et al., 2011 findings of "a group of neurons that inhibits inspiration" during SLN stim, but don't mention that it is the BotC and that the paper shows that swallow apnea is dependent on BotC. That is also supported by the Toor study. I don't understand how post-I (aka E) can be discussed without discussion of the BotC - this is a glaring omission.
• Why is it necessary for PiCo to innervate the cNTS? That is true if the conjecture that PiCo gates swallowing is true, as the cNTS is the only known region for central swallow gating. However, PiCo could influence afferent input to the NTS less directly, and therefore not function as a gating hub per se. The experimental evidence does not warrant the claim that PiCo gates swallowing. The definition of a swallow gate(s) is a topic of much debate and no conclusive experimental evidence has emerged for swallow gating regions to exist anywhere except in the NTS. The current study's evidence also does not meet the criteria necessary to conclusively call PiCo a swallow gate. The authors should soften this claim and language throughout the manuscript.
It is also unclear that PiCo acts directly on the swallow pattern generator to gate swallowing. It is not just "conceivable that the gating mechanism involves" the pons, but nearly certain. Swallow gating by respiratory activity may not be able to be ascribed to one particular location. At a minimum, it likely involves the NTS/DSG, pons, and possibly IRt (inclusive of PiCo). The authors are correct that "further studies are necessary to understand the interaction between PiCo and the pontine respiratory group on the gating swallow and other airway protective behaviors." This is why it shouldn't be stated that "this small brainstem microcircuits acts as a central gating mechanism for airway protective behaviors."
PiCo is likely part of the VSG (and thus the swallow pattern generator). PiCo, as part of the IRt/VSG could indeed be surveilling afferent information and providing output that affects swallow or other laryngeal activation and the coordination of these behaviors with breathing. However, this is not the responsibility of PiCo alone. This role is likely shared by other parts of the SPG, and may require distributed SPG network participation to be functional. If one were to stim other regions of the distributed SPG, similar results might be expected. When Toor et al silenced the PiCo area (and locations that lie at least lightly beyond the borders of what the present study defines as PiCo), stim-evoked fictive swallows were greatly suppressed. However, swallow-related apnea was unaffected. This supports the role of PiCo as a premotor relay for swallow motor activation, but not as the site that terminates inspiration. Therefore, it cannot be called a gate.• Similarly, Fig 7 does not accurately depict things that are already well-supported by evidence. PiCo should be included as part of the swallow pattern generator (VSG), not as a separate entity acting on it. The BotC and pons are glaring omissions. This study has not demonstrated the labeled inhibitory connection from DSG to PreBotC. The legend states speculations as fact and needs to be dialed way back to either include statements with solid experimental evidence or to clearly mark things as putative/speculative.
• The discussion of expiratory laryngeal motoneurons needs to be expanded and integrated better into the discussion of swallow, post-I, and other laryngeal motor activation. Why can't PiCo just be premotor to ELMs?
• Concerning the discussion of "PiCo's influence as a gate for airway protective behaviors is blurred...": The incomplete swallow motor sequence didn't seem super different in timing or duration compared to the fully transfected animals (comparing plots from Fig 6 to Fig S1, and Table S2 to Table S3. The values for swallow durations (XII and X) for each group for water and opto seem within similar ranges, as do the differences between water & opto-evoked swallows between strains. While the motor pattern is distinctive from the normal swallow, with laryngeal activity rather than submental activity leading, one might not even be able to call that a swallow. Is it evidence against a classic all-or-nothing swallow behavior any more than the graded swallow results from (fully transfected) Table S1?
• Please expand on this point and put it into context with others' results: "This brings into question whether this is the first evidence against the classic dogma of swallow as an "all or nothing" behavior, and/or whether this is an indication that activating the cholinergic/glutamatergic neurons in PiCo is not only gating the SPG, but is actually involved in assembling the swallow motor pattern itself."
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Reviewer #2 (Public Review):
The study "Postinspiratory complex acts as a gating mechanism regulating swallow-breathing coordination and other laryngeal behaviors" by Huff et al., provides additional insight into the role of the recently discovered postinspiratory complex during swallow-breathing coordination. The authors used optogenetics in mice to show that activation of the PiCo during inspiration or at the start of post-inspiration can evoke swallowing. At later stages of expiration, PiCo activation activates undefined laryngeal activities. The analysis of respiratory phase reset leads to the conclusion that the PiCo is important for central gating of swallow. In conclusion, the authors claim that swallow-breathing coordination depends on a defined microcircuit compromising the PiCo and the pre-Botzinger complex.
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Reviewer #3 (Public Review):
Huff et.al further characterise the anatomy and function of a population of excitatory medullary neurons, the Post-inspiratory Complex (PiCo), which they first described in 2016 as the origin of the laryngeal adduction that occurs in the post-inspiratory phase of quiet breathing. They propose an additional role for the glutamatergic and cholinergic PiCo interneurons in coordinating swallowing and protective airway reflexes with breathing, a critical function of the central respiratory apparatus, the neural mechanics of which have remained enigmatic. Using single allelic and intersectional allelic recombinase transgenic approaches, Huff et al. selectively excited choline acetyltransferase (ChAT) and vesicular glutamate transporter-2 (VGluT2) expressing neurons in the intermediate reticular nucleus of …
Reviewer #3 (Public Review):
Huff et.al further characterise the anatomy and function of a population of excitatory medullary neurons, the Post-inspiratory Complex (PiCo), which they first described in 2016 as the origin of the laryngeal adduction that occurs in the post-inspiratory phase of quiet breathing. They propose an additional role for the glutamatergic and cholinergic PiCo interneurons in coordinating swallowing and protective airway reflexes with breathing, a critical function of the central respiratory apparatus, the neural mechanics of which have remained enigmatic. Using single allelic and intersectional allelic recombinase transgenic approaches, Huff et al. selectively excited choline acetyltransferase (ChAT) and vesicular glutamate transporter-2 (VGluT2) expressing neurons in the intermediate reticular nucleus of anesthetised mice using an optogenetic approach, evoking a stereotyped swallowing motor pattern (indistinguishable from a water-induced swallow) during the early phase of the breathing cycle (within the first 10% of the cycle) or tonic laryngeal adduction (which tracked tetanically with stimulus length) during the later phase of the breathing cycle (after 70% of the cycle).
They further refine the anatomical demarcation of the PiCo using a combination of ChAT immunohistochemistry and an intersectional transgenic strategy by which fluorescent reporter expression (tdTomato) is regulated by a combinatorial flippase and cre recombinase-dependent cassette in triple allelic mice (Vglut2-ires2-FLPO; ChAT-ires-cre; Ai65).
Lastly, they demonstrate that the PiCo is anatomically positioned to influence the induction of swallowing through a series of neuroanatomical experiments in which the retrograde tracer Cholera Toxin B (CTB) was transported from the proposed location of the putative swallowing pattern generator within the caudal nucleus of the solitary tract (NTS) to glutamatergic ChAT neurons located within the PiCo.
Methods and Results
The experimental approach is appropriate and at the cutting edge for the field: advanced neuroscience techniques for neuronal stimulation (virally driven opsin expression within a genetically intersecting subset of neurons) applied within a sophisticated in vivo preparation in the anaesthetized mouse with electrophysiological recordings from functionally discrete respiratory and swallowing muscles. This approach permits selective stimulation of target cell types and simultaneous assessment of gain-of-function on multiple respiratory and swallowing outputs. This intersectional method ensures PiCo activation occurs in isolation from surrounding glutamatergic IRt interneurons, which serve a diverse range of homeostatic and locomotor functions, and immediately adjacent cholinergic laryngeal motor neurons within the nucleus ambiguous (seen by some as a limitation of the original study that first described the PiCo and its roll in post-I rhythm generation Anderson et al., 2016 Nature 536, 76-80). These experiments are technically demanding and have been expertly performed.The supplemental tracing experiments are of a lower standard. CTB is a robust retrograde tracer with some inherent limitations, paramount of which is the inadvertent labelling of neurons whose axons pass through the site of tracer deposition, commonly leading to false positives. In the context of labelling promiscuity by CTB, the small number of PiCo neurons labelled from the NTS (maybe 5 or 6 at most in an optical plane that features 20 or more PiCo neurons) is a concern. Even assuming that only a small subset of PiCo neurons makes this connection with the presumed swallowing CPG within the cNTS, interpretation is not helped by the low contrast of the tracer labelling (relative to the background) and the poor quality of the image itself. The connection the authors are trying to demonstrate between PiCo and the cNTS could be solidified using anterograde tracing data the authors should already have at hand (i.e. EYFP labelling driven by the con-fon AAV vectors from PiCo neurons (shown in Fig5), which should robustly label any projections to the cNTS).
The retrograde labelling from laryngeal muscles seems unnecessary: the laryngeal motor pool is well established (within the nAmb and ventral medulla), and it would be unprecedented for a population of glutamatergic neurons to form direct connections with muscles (beyond the sensory pool).
The authors support their claim that PiCo neurons gate laryngeal activity with breathing through the demonstration that selective activation of glutamatergic and cholinergic PiCo neurons is sufficient to drive oral/pharyngeal/laryngeal motor responses under anaesthesia and that such responses are qualitatively shaped by the phase of the respiratory cycle within which stimulation occurs. Optical stimulation within the first 10% of the respiratory cycle was sufficient to evoke a complete, stereotyped swallow that reset the breathing cycle, while stimuli within the later 70% of the cycle, evoked discharge of the laryngeal muscles in a stimulus length-dependent manner. Induced swallows were qualitatively indistinguishable from naturalistic swallow induced by the introduction of water into the oral cavity. The authors note that a detailed interpretation of induced laryngeal activity is probably beyond the technical limits of their recordings, but they speculate that this activity may represent the laryngeal adductor reflex. This seems like a reasonable conclusion.
The authors propose a model whereby the PiCo impinges upon the swallowing CPG (itself a poorly resolved structure) to explain their physiological data. The anatomical data presented in this study (retrograde transport of CTB from cNTS to PiCo) are insufficient to support this claim. As suggested above, complementary, high-quality, anterograde tracing data demonstrating connectivity between these structures as well as other brain regions would help to support this claim and broaden the impact of the study.
This study proposes that the PiCo in addition to serving as the site of generation of the post-I rhythm also gates swallowing and respiration. The scope of the study is small, and limited to the subfields of swallowing and respiratory neuroscience, however, this is an important basic biological question within these fields. The basic biological mechanisms that link these two behaviors, breathing and swallowing, are elusive and are critical in understanding how the brain achieves robust regulation of motor patterning of the aerodigestive tract, a mechanism that prevents aspiration of food and drink during ingestion. This study pushes the PiCo as a key candidate and supports this claim with solid functional data. A more comprehensive study demonstrating the necessity of the PiCo for swallow/breathing coordination through loss of function experiments (inhibitory optogenetics applied in the same transgenic context) along with robust connectivity data would solidify this claim.
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