An open-source tool for automated analysis of breathing behaviors in common marmosets and rodents
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Evaluation Summary:
This manuscript from Bishop et al aims to quantify the hypoxic and hyperoxic ventilatory response in the marmoset, an increasingly more common primate research model. The strongest contribution of the paper is the presentation of an analysis toolkit to perform unsupervised analyses of respiratory data, which are not widely available.
(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. The reviewers remained anonymous to the authors.)
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Abstract
The respiratory system maintains homeostatic levels of oxygen (O 2 ) and carbon dioxide (CO 2 ) in the body through rapid and efficient regulation of breathing frequency and depth (tidal volume). The commonly used methods of analyzing breathing data in behaving experimental animals are usually subjective, laborious, and time-consuming. To overcome these hurdles, we optimized an analysis toolkit for the unsupervised study of respiratory activities in animal subjects. Using this tool, we analyzed breathing behaviors of the common marmoset ( Callithrix jacchus ), a New World non-human primate model. Using whole-body plethysmography in room air as well as acute hypoxic (10% O 2 ) and hypercapnic (6% CO 2 ) conditions, we describe breathing behaviors in awake, freely behaving marmosets. Our data indicate that marmosets’ exposure to acute hypoxia decreased metabolic rate and increased sigh rate. However, the hypoxic condition did not augment ventilation. Hypercapnia, on the other hand, increased both the frequency and depth (i.e., tidal volume) of breathing.
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Author Response
Reviewer #1 (Public Review):
In this manuscript, Bishop et al. aim to quantify the ventilatory response to hypoxia and hypercapnia in the common marmoset, an increasingly more common primate research model. They also present an unsupervised analysis tool to quantify ventilatory behavior, which is a potentially major contribution to the respiratory field.
Strengths of this manuscript include the inclusion of male and female animals and the development of an analysis toolkit that may be less impacted by biases that are introduced when hand analyzing respiratory behavior, as is commonly done in the field. This tool could be of tremendous value to the respiratory community. Identification of sniffs, sighs, and apneas are often plagued by the qualitative nature of the analysis.
We thank the reviewer for taking their …
Author Response
Reviewer #1 (Public Review):
In this manuscript, Bishop et al. aim to quantify the ventilatory response to hypoxia and hypercapnia in the common marmoset, an increasingly more common primate research model. They also present an unsupervised analysis tool to quantify ventilatory behavior, which is a potentially major contribution to the respiratory field.
Strengths of this manuscript include the inclusion of male and female animals and the development of an analysis toolkit that may be less impacted by biases that are introduced when hand analyzing respiratory behavior, as is commonly done in the field. This tool could be of tremendous value to the respiratory community. Identification of sniffs, sighs, and apneas are often plagued by the qualitative nature of the analysis.
We thank the reviewer for taking their time to evaluate our submission and for the overall positive assessment of our submission.
Limitations of the study relate to the measure of the hypoxic and hypercapnic ventilatory drive. Tidal volume in whole body plethysmography is not accurate unless the plethysmograph and body temperature are taken into account. (See, https://pubmed.ncbi.nlm.nih.gov/25080926/). This is particularly important when the animal's core body temperature changes during hypoxia because of a fall in metabolic rate. The decrease in VCO2 shown here suggests that this is occurring here.
We thank the reviewer for their comment. We applied acute hypoxia and hypercapnia to perturb breathing behaviors and used our analysis tool to evaluate said disturbed respiratory behaviors. We have addressed the limitations of our studies in the revised submission. In addition, and because of this limitation, we include an arbitrary unit (a.u.) for tidal volume (and other characteristics of breathing derived from tidal volume).
It is worth pointing out that the fall in VCO2 is not typically observed in humans. So, while the authors conclude that minute ventilation does not increase in the marmoset, it is not necessarily a valid conclusion that that hypoxia ventilatory drive is low because VE should be expressed as a function of VCO2. If VCO2 falls but VE is constantly, ventilation per unit metabolism will actually have increased. Ventilation may also be underestimated here because of the fall in core body temp that likely coincides with a lower VCO2.
We thank the reviewer for this comment. The data on changes of metabolic rate (by measuring VCO2 or VO2) during hypoxia in human subjects are not consistent (for instance see PMID: 2390141, Figure 3 clearly shows a decrease of ~50% in metabolic rate during hypoxia). Therefore, we have soften the language in our submitted revision.
In addition, in the revised manuscript, we have performed the recommended analysis to express VE as a function of VCO2. However, hypoxia did not increase the ventilation efficiency (VE/VCO2) in marmosets. We have added the new data (Figure 4H) and discussed it in the revised manuscript.
It is also worth noting that the hypoxic ventilatory response is not necessarily linear and the full range of the response is not characterized. For example, 15% O2 in the rat elicits very little response but there is a robust response with 9% O2. It is also worth noting, relevant to the previous points, that this is not an isocapnic ventilatory response, so the hypoxic response is certainly confounded by the changing CO2 which may not mimic situations like sleep apnea.
We thank the reviewer for this comment. In the revised manuscript, we added that we have applied ‘acute’ hypoxic/hypercapnic challenges and discussed the limitation of our study.
Reviewer #2 (Public Review):
I do not see any fundamental flaws in it as such.
However, what really compromises the paper, it the lack of a "punch line". It is highly descriptive rather than analytical, it reads like a list of mostly predictable outcomes, but what is the question, what is the novelty, why is it important... This does not come out at all. On one hand it is important to have such basic information about marmosets but is it best placed into a non-specialist journal? In addition, the whole point of getting involved with monkeys is because they are closer to humans than rodents, but authors did not fully explore these similarities/differences or focus on them or try to explain them. One would want to have a clear conclusion in the end, how closely they resemble humans, for what type of experiments they are better than rodents, because of what... But this is not evident. Neither is it clear what is the value of the novel protocol for data analysis which seems to have been a major effort. In the end we are left with the impression that the results you get with it are the same as with the old protocols... What is its value then? Something needs to be done to make this paper attract readers others but only specifically interested in this topic.We thank the reviewer for these comments. We acknowledge that the initial submission was not as clear as we had hoped. We have revised the manuscript and added more details about our new analysis tool and further strengthened its applicability by including new analysis from a rodent model. We believe the major contribution of this manuscript to the field is providing a new open-source tool to analyze complex breathing behavior signals in conscious, awake, and active laboratory animals. In this manuscript, we demonstrate the strength of this approach in rapidly expediting analysis of breathing behaviors, which we analyze of the common marmoset and rat, yet could be equally applicable to other animal models.
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Evaluation Summary:
This manuscript from Bishop et al aims to quantify the hypoxic and hyperoxic ventilatory response in the marmoset, an increasingly more common primate research model. The strongest contribution of the paper is the presentation of an analysis toolkit to perform unsupervised analyses of respiratory data, which are not widely available.
(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. The reviewers remained anonymous to the authors.)
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Reviewer #1 (Public Review):
In this manuscript, Bishop et al. aim to quantify the ventilatory response to hypoxia and hypercapnia in the common marmoset, an increasingly more common primate research model. They also present an unsupervised analysis tool to quantify ventilatory behavior, which is a potentially major contribution to the respiratory field.
Strengths of this manuscript include the inclusion of male and female animals and the development of an analysis toolkit that may be less impacted by biases that are introduced when hand analyzing respiratory behavior, as is commonly done in the field. This tool could be of tremendous value to the respiratory community. Identification of sniffs, sighs, and apneas are often plagued by the qualitative nature of the analysis.
Limitations of the study relate to the measure of the hypoxic …
Reviewer #1 (Public Review):
In this manuscript, Bishop et al. aim to quantify the ventilatory response to hypoxia and hypercapnia in the common marmoset, an increasingly more common primate research model. They also present an unsupervised analysis tool to quantify ventilatory behavior, which is a potentially major contribution to the respiratory field.
Strengths of this manuscript include the inclusion of male and female animals and the development of an analysis toolkit that may be less impacted by biases that are introduced when hand analyzing respiratory behavior, as is commonly done in the field. This tool could be of tremendous value to the respiratory community. Identification of sniffs, sighs, and apneas are often plagued by the qualitative nature of the analysis.
Limitations of the study relate to the measure of the hypoxic and hypercapnia ventilatory drive. Tidal volume in whole body plethysmography is not accurate unless the plethysmograph and body temperature are taken into account. (See, https://pubmed.ncbi.nlm.nih.gov/25080926/). This is particularly important when the animal's core body temperature changes during hypoxia because of a fall in metabolic rate. The decrease in VCO2 shown here suggests that this is occurring here.
It is worth pointing out that the fall in VCO2 is not typically observed in humans. So, while the authors conclude that minute ventilation does not increase in the marmoset, it is not necessarily a valid conclusion that that hypoxia ventilatory drive is low because VE should be expressed as a function of VCO2. If VCO2 falls but VE is constantly, ventilation per unit metabolism will actually have increased. Ventilation may also be under-estimated here because of the fall in core body temp that likely coincides with a lower VCO2.
It is also worth noting that the hypoxic ventilatory response is not necessarily linear and the full range of the response is not characterized. For example, 15% O2 in the rat elicits very little response but there is a robust response with 9% O2. It is also worth noting, relevant to the previous points, that this is not an isocapnic ventilatory response, so the hypoxic response is certainly confounded by the changing CO2 which may not mimic situations like sleep apnea.
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Reviewer #2 (Public Review):
I do not see any fundamental flaws in this paper as such. However, what really compromises the paper, it the lack of a "punch line". It is highly descriptive rather than analytical, it reads like a list of mostly predictable outcomes, but what is the question, what is the novelty, why is it important... This does not come out at all. On one hand it is important to have such basic information about marmosets but is it best placed into a non-specialist journal? In addition, the whole point of getting involved with monkeys is because they are closer to humans than rodents, but authors did not fully explore these similarities/differences or focus on them or try to explain them. One would want to have a clear conclusion in the end, how closely they resemble humans, for what type of experiments they are better …
Reviewer #2 (Public Review):
I do not see any fundamental flaws in this paper as such. However, what really compromises the paper, it the lack of a "punch line". It is highly descriptive rather than analytical, it reads like a list of mostly predictable outcomes, but what is the question, what is the novelty, why is it important... This does not come out at all. On one hand it is important to have such basic information about marmosets but is it best placed into a non-specialist journal? In addition, the whole point of getting involved with monkeys is because they are closer to humans than rodents, but authors did not fully explore these similarities/differences or focus on them or try to explain them. One would want to have a clear conclusion in the end, how closely they resemble humans, for what type of experiments they are better than rodents, because of what... But this is not evident.
Neither is it clear what is the value of the novel protocol for data analysis which seems to have been a major effort. In the end we are left with the impression that the results you get with it are the same as with the old protocols... What is its value then? Something needs to be done to make this paper attract readers others but only specifically interested in this topic.
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