Predictions and experimental tests of a new biophysical model of the mammalian respiratory oscillator

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1. 

   Version published to 10.7554/elife.74762 on eLife

   Jul 7, 2022
2. 

   eLife

   Mar 23, 2022

   Author Response:

   Reviewer #1:

   The present work by Phillips et al., builds on a previously published (eLife 2019, 8:e41555) computational model that showed how rhythmicity and the amplitude of respiratory oscillations involve distinct biophysical mechanisms. In particular, the model predicts that respiratory rhythm can be independent of calcium-activated non-selective cation current activation, and that this determines population activity amplitude. In contrast, rhythm depends on sodium currents in a subpopulation of cells forming a preBötC rhythmogenic kernel. The past model proposed by Phillips et al., (2019) consistently reproduced some previously published experimental studies.

   The experimental data obtained in this current work systematically demonstrate that some of the simulations and predictions generated from their …

   Author Response:

   Reviewer #1:

   The present work by Phillips et al., builds on a previously published (eLife 2019, 8:e41555) computational model that showed how rhythmicity and the amplitude of respiratory oscillations involve distinct biophysical mechanisms. In particular, the model predicts that respiratory rhythm can be independent of calcium-activated non-selective cation current activation, and that this determines population activity amplitude. In contrast, rhythm depends on sodium currents in a subpopulation of cells forming a preBötC rhythmogenic kernel. The past model proposed by Phillips et al., (2019) consistently reproduced some previously published experimental studies.

   The experimental data obtained in this current work systematically demonstrate that some of the simulations and predictions generated from their computational model are accurate, thereby illustrating the robustness of their computational model.

   Strengths:

   Both the computational model and empirical data provided in this work further foster our understanding on how the preBötC generates (respiratory/inspiratory) rhythmogenesis and highlights the existence of distinct biophysical mechanisms involved in rhythmicity and the amplitude of respiratory oscillations. Collectively, this work is of great interest to the respiratory neuroscientist community.

   Weaknesses:

   Whereas the major claims of this work are supported by solid experimental data, the manuscript is written in a highly technical manner that is not comprehensible for scientists not familiar with computational modeling and electrophysiology. It would be desirable that the authors could make the text more accessible to a larger audience.

   While we cannot avoid presenting all of the technical details of our study, in this revised manuscript we have made sure that the significance statements in the Introduction and Discussion are presented in a manner that makes the essential results of our study accessible to a general audience.

   Reviewer #2:

   In this manuscript, Phillips et al. address the relevance of the persistent inward conductances, INaP and ICAN, for inspiratory rhythm and pattern generation. The authors previously developed a computational model of the inspiratory rhythm generator, the preBötzinger Complex (preBötC), that relied on INaP for rhythm generation and ICAN for pattern generation. Here, they perform experiments designed to test certain predictions of their model using thin rhythmic medullary slices from triple transgenic mice where both tdTomato and ChR2-EYFP are expressed in glutamatergic VGLUT2-expressing neurons. The authors show that pharmacological blockade of INaP leads to dose-dependent decreases in burst frequency and amplitude under baseline conditions and at varying levels of tonic optogenetic excitation with high concentrations of the blocker preventing rhythmic bursting even at high laser powers. Pharmacological blockade of ICAN reduces amplitude, but does not significantly affect frequency at baseline and causes an increase in frequency when laser power is increased. The authors make the claim that these data support their model and the hypothesis that INaP is essential for preBötC rhythmogenesis and ICAN is essential for determining burst amplitude, but is dispensable for rhythm generation.

   The strengths of the manuscript are that the computational model is revised to include a biophysical model for channelrhodopsin and that the modeling and experiments support the proposed role for ICAN in burst generation. The prediction of an increase in frequency with increased tonic excitation when ICAN is blocked is of particular interest.

   Despite these strengths, a number of major issues significantly weaken the manuscript and limit its impact in advancing understanding of rhythm and pattern generation in preBötC.

   1. Optogenetic stimulation. The authors use an optogenetic approach that may be more complex than assumed and that is not adequately validated in their model. The transgenic mouse used expresses both tdTomato and ChR2-EYFP in all glutamatergic VGLUT2-expressing neurons. The authors assume that bilateral illumination over preBötC enables depolarization specifically in the preBötC excitatory population. However, ChR2 will be expressed in all glutamatergic neurons, so the illumination may depolarize terminals or fibers of passage from glutamatergic neurons outside the preBötC (even those whose somata were removed in slicing). Illumination of non-rhythmogenic preBötC neurons may affect interpretation of their results and congruence with their model, which only contains the preBötC rhythmogenic population. Furthermore, ChR2-induced depolarization may interact unexpectedly with other membrane properties and conductances. Two examples that indicate that the optogenetic stimulation protocol may not be straightforward is the 1-3 minute inhibition of rhythmicity following illumination (p 8, line 20-22, Figure 3B) and what appears to be a hyperpolarization following 5 mW illumination in their whole cell patch clamp recordings (Fig 2C). While the voltage dependence of ChR2 in their model is presented, whether these other phenomena are also reproduced in their model is not demonstrated, calling into question how to interpret their comparisons of experimental and model results.

   We thank the reviewer for this comment about identifying potential sources of error, off-target effects, and experimental limitations that need to be considered when interpreting our experimental results. Discussion of the potential off-target ChR2 expression is now included, following the discussion of possible non-uniform ChR2 expression and/or activation.

   1. Challenges to the INaP hypothesis in published results. The biggest issue with the manuscript is its central hypothesis that INaP is essential for rhythmogenesis. This hypothesis has faced considerable scrutiny, and a number of papers appear to invalidate a necessary role for INaP in preBötC rhythmogenesis.

   The discussion has been updated to provide a more balanced and detailed discussion of these issues. See the “Previous pharmacological studies and proposed roles of INaP in preBötC inspiratory network rhythm generation” subsection of the Discussion. Briefly, the current study is a direct test of the hypothesis presented in Pace et al. (2007). If, as suggested by Pace et al., bath application of TTX or RZ impacts the inspiratory rhythm by reducing preBötC excitability rather than by affecting the essential mechanism(s) of rhythm generation, then increasing preBötC excitability via optogenetic stimulation should restart the rhythm even after complete INaP blockade. Our results show that, to the contrary, the preBötC is incapable of generating rhythmic output after complete INaP block even under optogenetic stimulation (Figures 4 and 5), demonstrating that INaP is essential for preBötC rhythm generation in this reduced in vitro preparation.

   The authors mention these other results superficially in the Discussion, but do not grapple with their clear challenges to the INaP hypothesis. Pace et al. (2007) showed that bilateral microinjection of riluzole or low concentrations of TTX into preBötC failed to stop the rhythm and that the pharmacological effects of these blockers could be explained by their effects on raphe excitability, which provides tonic excitatory drive to the preBötC. The authors propose that these conflicting results can be explained by differences in slice thickness and incomplete pharmacological penetration; however, the Pace paper specifically addressed this issue by microinjecting the drugs 100 um below the surface. Further, the raphe microinjection provide an alternative experimentally-validated explanation for many prior and current pharmacological experiments involving INaP blockers. All blockers were bath-applied here, and these concerns were not addressed experimentally.

   Here, we propose that the failure of bilateral microinjection of RZ and TTX to abolish preBötC inspiratory rhythm generation is most likely due to incomplete spread of these pharmacological agents across the inspiratory rhythmogenic circuitry due to the thick slice preparations used in Pace et al. (2007). Even though Pace et al. (2007) attempted to overcome this issue by directly microinjecting TTX and RZ directly into the preBötC, in thick slices effective drug penetration and diffusion may still be an issue. Moreover, the results of Pace et al. (2007) have not been reproduced and in fact have been refuted in a follow up study by Koizumi and Smith (2008) utilizing thinner slices. A detailed discussion of these points has been added.

   Finally, off-target effects of INaP blockers, particularly at the higher concentrations, were also not addressed.

   To the best of our knowledge only RZ, not TTX, potentially produces notable off-target effects at the concentrations used in this study (≤20µM RZ, ≤20 nM TTX)). At this concentration, the primary off-target effect of RZ can be attenuation of excitatory synaptic transmission as discussed in the manuscript. Previous computational simulations (Phillips and Rubin, 2019) showed that attenuation of excitatory synaptic transmission was required to explain the slightly larger decrease in preBötC burst amplitude seen with RZ (compared to TTX) microinjection into the preBötC in thin in vitro slice preparations (Koizumi and Smith, 2008). Importantly, off-target attenuation of excitatory synaptic transmission was incorporated into the current study based on the findings presented in Phillips and Rubin, 2019. Like the previous study, we found that a 20-25% reduction in the weights of excitatory synapses was required in order to produce the slightly larger downward shift in the amplitude vs laser power curves seen with 5µM and 10µM riluzole vs TTX block of INaP.

   Moreover, although TTX is generally associated with blockade of the fast action potential generating Na+ current, the low concentrations used in this study have previously been shown to not affect this current or action potential generation, see Koizumi and Smith (2008). The same is true in the current study, as action potential generation does not appear to be affected by the concentrations of TTX or RZ used here (see Figures 4 & 5).

   Importantly, even if the off-target effects of TTX and RZ application contribute to the experimental results characterized in this study, these pharmacological agents still compromise the fundamental mechanism(s) of rhythm generation within the preBötC, which is inconsistent with the primary conclusion reached by Pace et al. (2007). This is demonstrated by the fact that the preBötC is not capable of generating a rhythm following TTX or riluzole application at concentrations ≥20 nM or 20 µM, respectively.

   In addition to INaP blockers, other published results show that rhythmicity can occur without high frequency bursts necessary for INaP activation, and pharmacology experiments in situ also suggest that rhythmicity can persist in more intact networks without INaP. These issues are discussed but not addressed experimentally. Thus, the substantial body of experimental work that is inconsistent with the INaP hypothesis remains relevant.

   The experimental observation that INaP may not be necessary for respiratory rhythm generation in more intact preparations is now discussed in detail. Understanding how this preBötC model interacts in simulations representing a more intact preparation by incorporating neuronal subtypes of the Bötzinger Complex involved in respiratory pattern or inputs from other respiratory nuclei such as the Kölliker-Fuse nucleus, parabrachial nucleus, retrotrapezoid nucleus or other higher brainstem regions known to impact breathing are important. However, computational and experimental analyses addressing these issues have been previously performed (e.g., Smith et al., 2007; Phillips and Rubin, 2019) and extensions of these analyses are beyond the scope of the current study and is therefore left for future investigation.

   1. Model limitations. Experimental confirmation of a limited set of predictions of a reduced model does not strongly support a particular model mechanism if the model does not include known conductances/properties of the biological system and does not reproduce other experimentally observed phenomena. Without including burst-terminating conductances, physiological connectivity and synaptic properties, and perhaps other preBötC populations, e.g., inhibitory neurons, the experimental results may not uniquely validate the model. Additionally, the model should be capable of reproducing a variety of experimentally observed preBötC phenomenology besides those directly related to INaP and ICAN. Without such constraints, the model could easily be tuned (and is in fact tuned in this manuscript) to reproduce selected results, severely limiting the validity and generalizability of the model and its mechanisms.

   As noted by this reviewer, the current model has some limitations that have the potential to impact the model’s behavior and potentially the model’s predictions. The limitations and assumptions of the current model are discussed in detail in the “Extensions and limitations of the model” section of the Discussion. Importantly, the model omits some additional biophysical mechanisms that may augment/shape inspiratory bursting and account for some differences in the preBötC behavior in the model and experiment, such as the post-stimulation decrease in preBötC network burst frequency. This limitation is discussed in detail.

   Although we agree with this reviewer about the importance of understanding model limitations, it is also important to point out that all computational models of biological systems are massively simplified compared to the reality of the biological system being investigated and require some degree of “tuning”. Omission of critical factors does have the potential to limit the validity and generalizability of a model and its proposed mechanism(s). However, such simplification does not eliminate the utility of computational modeling. Despite their inherent simplification, computational models are essential for our understanding of neuronal dynamics, as they provide a way of formalizing and quantifying otherwise vague concepts such as the mechanisms proposed to underlie inspiratory rhythm and pattern generation. Perhaps the most useful aspect of computational modeling is the ability to generate experimentally testable and mechanism-specific predictions that would be difficult or impossible to generate through intuition alone (see Marder eLife 2020;9:e60703 DOI: 10.7554/eLife.60703). Importantly, the mechanism-specific model predictions (Figure 1) that motivated the current study likely could not have been generated without the insights provided by computational modeling. Moreover, these initial predictions were made without any modification to the tuning of the initial model presented in Phillips et al., 2019 and the predicted directional shifts with blocking INaP and ICAN in the preBötC burst frequency/amplitude vs network depolarization were made in preliminary simulations prior to any comparable experimental measurements (see Phillips 2017). Future studies will undoubtedly identify shortcomings of the current model, which will spur further development and refinement of our theoretical understanding of the biophysical mechanism(s) underlying inspiratory rhythm and pattern generation in the preBötC.

   1. Statistical comparisons. Statistical comparisons are relatively limited in this manuscript. Methods mention Student's t-test or the Wilcoxon signed rank test, but it appears that some of the data, e.g., frequency/amplitude dose dependent curves, downward shifts of frequency or amplitude in drug, and comparisons between model and experiment, would require parametric or non-parametric multiple comparison tests, such as ANOVA or Kolmogorov-Smirnov. Without such comparisons, qualitative descriptions may mask non-significant variations or statistically significant differences may be missed.

   As the reviewer suggested, we have re-analyzed statistical significance with non-parametric Wilcoxon matched-pairs signed rank test or Kolmogorov-Smirnov test when comparing two groups, and two-way ANOVA test for comparing multiple groups in conjunction with post hoc Tukey’s HSD test for pairwise comparison. We have updated the results section, and also methods section accordingly. Please note that these new statistical analyses did not change the experimental results in terms of significance.

   Read the original source
3. 

   eLife

   Mar 23, 2022

   Evaluation Summary:

   In this paper the authors test three hypotheses: 1) INap and ICAN blockade alter network excitability in the preBötC, the region of the brainstem that generates inspiratory breathing rhythm; 2) that INaP is essential for preBötC rhythmogenesis; 3) ICAN is essential for generating the amplitude of rhythmic output but not rhythm generation. They test these hypotheses using optogenetic manipulation of local preBötC excitability and the use of pharmacologic blockade of INaP and ICAN. This manuscript provides substantive evidence for the role of INaP in modulating breathing frequency and ICAN in altering amplitude with some interesting boundary conditions when ICAN and INaP are selectively blocked and tests predictions about these currents using computational simulation.

   (This preprint has been reviewed by eLife. We …

   Evaluation Summary:

   In this paper the authors test three hypotheses: 1) INap and ICAN blockade alter network excitability in the preBötC, the region of the brainstem that generates inspiratory breathing rhythm; 2) that INaP is essential for preBötC rhythmogenesis; 3) ICAN is essential for generating the amplitude of rhythmic output but not rhythm generation. They test these hypotheses using optogenetic manipulation of local preBötC excitability and the use of pharmacologic blockade of INaP and ICAN. This manuscript provides substantive evidence for the role of INaP in modulating breathing frequency and ICAN in altering amplitude with some interesting boundary conditions when ICAN and INaP are selectively blocked and tests predictions about these currents using computational simulation.

   (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #3 agreed to share their names with the authors.)

   Read the original source
4. 

   eLife

   Mar 23, 2022

   Reviewer #1 (Public Review):

   The present work by Phillips et al., builds on a previously published (eLife 2019, 8:e41555) computational model that showed how rhythmicity and the amplitude of respiratory oscillations involve distinct biophysical mechanisms. In particular, the model predicts that respiratory rhythm can be independent of calcium-activated non-selective cation current activation, and that this determines population activity amplitude. In contrast, rhythm depends on sodium currents in a subpopulation of cells forming a preBötC rhythmogenic kernel. The past model proposed by Phillips et al., (2019) consistently reproduced some previously published experimental studies.

   The experimental data obtained in this current work systematically demonstrate that some of the simulations and predictions generated from their computational …

   Reviewer #1 (Public Review):

   The present work by Phillips et al., builds on a previously published (eLife 2019, 8:e41555) computational model that showed how rhythmicity and the amplitude of respiratory oscillations involve distinct biophysical mechanisms. In particular, the model predicts that respiratory rhythm can be independent of calcium-activated non-selective cation current activation, and that this determines population activity amplitude. In contrast, rhythm depends on sodium currents in a subpopulation of cells forming a preBötC rhythmogenic kernel. The past model proposed by Phillips et al., (2019) consistently reproduced some previously published experimental studies.

   The experimental data obtained in this current work systematically demonstrate that some of the simulations and predictions generated from their computational model are accurate, thereby illustrating the robustness of their computational model.

   Strengths:

   Both the computational model and empirical data provided in this work further foster our understanding on how the preBötC generates (respiratory/inspiratory) rhythmogenesis and highlights the existence of distinct biophysical mechanisms involved in rhythmicity and the amplitude of respiratory oscillations. Collectively, this work is of great interest to the respiratory neuroscientist community.

   Weaknesses:

   Whereas the major claims of this work are supported by solid experimental data, the manuscript is written in a highly technical manner that is not comprehensible for scientists not familiar with computational modeling and electrophysiology. It would be desirable that the authors could make the text more accessible to a larger audience.

   Read the original source
5. 

   eLife

   Mar 23, 2022

   Reviewer #2 (Public Review):

   In this manuscript, Phillips et al. address the relevance of the persistent inward conductances, INaP and ICAN, for inspiratory rhythm and pattern generation. The authors previously developed a computational model of the inspiratory rhythm generator, the preBötzinger Complex (preBötC), that relied on INaP for rhythm generation and ICAN for pattern generation. Here, they perform experiments designed to test certain predictions of their model using thin rhythmic medullary slices from triple transgenic mice where both tdTomato and ChR2-EYFP are expressed in glutamatergic VGLUT2-expressing neurons. The authors show that pharmacological blockade of INaP leads to dose-dependent decreases in burst frequency and amplitude under baseline conditions and at varying levels of tonic optogenetic excitation with high …

   Reviewer #2 (Public Review):

   In this manuscript, Phillips et al. address the relevance of the persistent inward conductances, INaP and ICAN, for inspiratory rhythm and pattern generation. The authors previously developed a computational model of the inspiratory rhythm generator, the preBötzinger Complex (preBötC), that relied on INaP for rhythm generation and ICAN for pattern generation. Here, they perform experiments designed to test certain predictions of their model using thin rhythmic medullary slices from triple transgenic mice where both tdTomato and ChR2-EYFP are expressed in glutamatergic VGLUT2-expressing neurons. The authors show that pharmacological blockade of INaP leads to dose-dependent decreases in burst frequency and amplitude under baseline conditions and at varying levels of tonic optogenetic excitation with high concentrations of the blocker preventing rhythmic bursting even at high laser powers. Pharmacological blockade of ICAN reduces amplitude, but does not significantly affect frequency at baseline and causes an increase in frequency when laser power is increased. The authors make the claim that these data support their model and the hypothesis that INaP is essential for preBötC rhythmogenesis and ICAN is essential for determining burst amplitude, but is dispensable for rhythm generation.

   The strengths of the manuscript are that the computational model is revised to include a biophysical model for channelrhodopsin and that the modeling and experiments support the proposed role for ICAN in burst generation. The prediction of an increase in frequency with increased tonic excitation when ICAN is blocked is of particular interest.

   Despite these strengths, a number of major issues significantly weaken the manuscript and limit its impact in advancing understanding of rhythm and pattern generation in preBötC.

   1. Optogenetic stimulation. The authors use an optogenetic approach that may be more complex than assumed and that is not adequately validated in their model. The transgenic mouse used expresses both tdTomato and ChR2-EYFP in all glutamatergic VGLUT2-expressing neurons. The authors assume that bilateral illumination over preBötC enables depolarization specifically in the preBötC excitatory population. However, ChR2 will be expressed in all glutamatergic neurons, so the illumination may depolarize terminals or fibers of passage from glutamatergic neurons outside the preBötC (even those whose somata were removed in slicing). Illumination of non-rhythmogenic preBötC neurons may affect interpretation of their results and congruence with their model, which only contains the preBötC rhythmogenic population. Furthermore, ChR2-induced depolarization may interact unexpectedly with other membrane properties and conductances. Two examples that indicate that the optogenetic stimulation protocol may not be straightforward is the 1-3 minute inhibition of rhythmicity following illumination (p 8, line 20-22, Figure 3B) and what appears to be a hyperpolarization following 5 mW illumination in their whole cell patch clamp recordings (Fig 2C). While the voltage dependence of ChR2 in their model is presented, whether these other phenomena are also reproduced in their model is not demonstrated, calling into question how to interpret their comparisons of experimental and model results.

   2. Challenges to the INaP hypothesis in published results. The biggest issue with the manuscript is its central hypothesis that INaP is essential for rhythmogenesis. This hypothesis has faced considerable scrutiny, and a number of papers appear to invalidate a necessary role for INaP in preBötC rhythmogenesis. The authors mention these other results superficially in the Discussion, but do not grapple with their clear challenges to the INaP hypothesis. Pace et al. (2007) showed that bilateral microinjection of riluzole or low concentrations of TTX into preBötC failed to stop the rhythm and that the pharmacological effects of these blockers could be explained by their effects on raphe excitability, which provides tonic excitatory drive to the preBötC. The authors propose that these conflicting results can be explained by differences in slice thickness and incomplete pharmacological penetration; however, the Pace paper specifically addressed this issue by microinjecting the drugs 100 um below the surface. Further, the raphe microinjection provide an alternative experimentally-validated explanation for many prior and current pharmacological experiments involving INaP blockers. All blockers were bath-applied here, and these concerns were not addressed experimentally. Finally, off-target effects of INaP blockers, particularly at the higher concentrations, were also not addressed. In addition to INaP blockers, other published results show that rhythmicity can occur without high frequency bursts necessary for INaP activation, and pharmacology experiments in situ also suggest that rhythmicity can persist in more intact networks without INaP. These issues are discussed but not addressed experimentally. Thus, the substantial body of experimental work that is inconsistent with the INaP hypothesis remains relevant.

   3. Model limitations. Experimental confirmation of a limited set of predictions of a reduced model does not strongly support a particular model mechanism if the model does not include known conductances/properties of the biological system and does not reproduce other experimentally observed phenomena. Without including burst-terminating conductances, physiological connectivity and synaptic properties, and perhaps other preBötC populations, e.g., inhibitory neurons, the experimental results may not uniquely validate the model. Additionally, the model should be capable of reproducing a variety of experimentally observed preBötC phenomenology besides those directly related to INaP and ICAN. Without such constraints, the model could easily be tuned (and is in fact tuned in this manuscript) to reproduce selected results, severely limiting the validity and generalizability of the model and its mechanisms.

   4. Statistical comparisons. Statistical comparisons are relatively limited in this manuscript. Methods mention Student's t-test or the Wilcoxon signed rank test, but it appears that some of the data, e.g., frequency/amplitude dose dependent curves, downward shifts of frequency or amplitude in drug, and comparisons between model and experiment, would require parametric or non-parametric multiple comparison tests, such as ANOVA or Kolmogorov-Smirnov. Without such comparisons, qualitative descriptions may mask non-significant variations or statistically significant differences may be missed.

   Read the original source
6. 

   eLife

   Mar 23, 2022

   Reviewer #3 (Public Review):

   In "Predictions and experimental tests of a new biophysical model of the mammalian respiratory oscillator" the authors test three hypotheses: 1) INap and ICAN blockade alter network excitability in the preBötC, the region of the brainstem that generates inspiratory breathing rhythm; 2) that INaP is essential for preBötC rhythmogenesis; 3) ICAN is essential for generating the amplitude of rhythmic output but not rhythm generation. They test these hypotheses using optogenetic manipulation of local preBötC excitability and the use of pharmacologic blockade of INaP and ICAN.

   The manuscript is well-written and clear. The experiments are appropriate to test the hypotheses and the data is convincing. This manuscript is significant because it provides substantive evidence for the role of INaP in modulating breathing …

   Reviewer #3 (Public Review):

   In "Predictions and experimental tests of a new biophysical model of the mammalian respiratory oscillator" the authors test three hypotheses: 1) INap and ICAN blockade alter network excitability in the preBötC, the region of the brainstem that generates inspiratory breathing rhythm; 2) that INaP is essential for preBötC rhythmogenesis; 3) ICAN is essential for generating the amplitude of rhythmic output but not rhythm generation. They test these hypotheses using optogenetic manipulation of local preBötC excitability and the use of pharmacologic blockade of INaP and ICAN.

   The manuscript is well-written and clear. The experiments are appropriate to test the hypotheses and the data is convincing. This manuscript is significant because it provides substantive evidence for the role of INaP in modulating breathing frequency and ICAN in altering amplitude with some interesting boundary conditions when ICAN and INaP are selectively blocked. Of particular value is the addition of a channelrhodopsin current (based on a Markov formalism) to the authors' previously published model.

   The authors provide strong evidence testing their hypotheses and showing the importance of both INaP and ICAN for the generation of reliable breathing rhythm.

   The results presented here provide strong evidence for separate roles for INaP and ICAN in modulating frequency and amplitude of central respiratory drive.

   Read the original source
7. 

   Version published to 10.1101/2021.10.29.466442v1 on bioRxiv

   Nov 1, 2021