On 2017 Jun 05, Frances Cheng commented:

Immobility in mice, calm or stressed?

Frances Cheng, PhD; Ingrid Taylor, DVM; Emily R Trunnell, PhD

People for the Ethical Treatment of Animals

This paper by Yackle, et al. claims to have found a link between breathing and calmness in mice. However, the conclusions made by the authors leave several critical questions unanswered.

Mice are naturally inquisitive and the expression of exploratory behavior is generally interpreted as good welfare. Conversely, being motionless or less exploratory is typically thought to be indicative of stress, pain, or poor welfare. There is no established relationship between calmness and sitting still; in fact the literature would likely attribute time spent immobile to anxiety in this species. Similarly, the relationship between grooming and calmness is not clear. Grooming can be elicited by both stressful and relaxing situations, and as such is problematic to use as an absolute marker of stress levels. In some cases, grooming can be a stress reliever and restraint stress can increase grooming (1). A study by Kaleuff and Tuohima attempted to differentiate between stress grooming and relaxed grooming, stating: “While a general pattern of self-grooming uninterrupted cephalocaudal progression is normally observed in no-stress (comfort) conditions in mice and other rodents, the percentage of ‘incorrect’ transitions between different stages and the percentage of interrupted grooming bouts may be used as behavioural marker of stress" (2). Indeed the preBötC ablated mouse in this supplementary video (http://science.sciencemag.org/content/sci/suppl/2017/03/29/355.6332.1411.DC1/aai7984s1.mp4) appears to be less active, but without other objective measurements, it is a leap to conclude that this mouse is calm.

The chamber used to measure the animals’ behavior is extremely small and inadequate for observing mouse behavior and making conclusions about calmness or other emotions, particularly when the differences in behavior between the two mice being compared are very subtle, as they are here. In addition, simply being in a chamber of this size could potentially be restraining and stress-inducing. A larger chamber would allow for more traditional measures of calmness and anxiety, such as exploratory behavior, where the amount of time the mouse spends along the wall of the chamber versus the amount of time he or she leaves the safety of the walls to explore the center area is measured and scored (the Open Field test).

As mentioned, sitting still does not necessarily dictate calmness, and in many behavioral paradigms, immobility is thought to be an outward sign of anxiety or distress. The authors mention that they observe different breathing rates associated with different behaviors, e.g., faster during sniffing and slower during grooming; however, observed breathing rate alone cannot be used as the sole measure to associate an emotion with a behavior. Consider that humans under stress can hyperventilate when sitting still. It would be more informative, and more applicable to calmness, to know whether or not ablation of Cdh9/Dbx1 double-positive preBötC neurons would influence one’s ability to control their breathing and potentially to breathe slower during a psychologically stressful situation, rather than how the ablation impacts breathing coupled with normal physiological functions.

It is not clear if the experimental group is physiologically capable of breathing faster in response to external stimuli. Not being able to do so—in other words being programmed to breathe a certain way—could be distressing. The lack of compensatory respiration mechanisms, such as increased respiration in unknown, potentially dangerous situations, could affect prey species such as mice in ways that have not been previously characterized.

The experimental groups were born with full use of the Cdh9/Dbx1 double-positive preBötC neurons. These neurons were ablated in adult animals. If these animals did not have a full range of control of their breathing after ablation, they might have experienced unpleasant psychological reactions to the forced change in breathing pattern, which could be distressing.

In the Methods section, the authors did not specify whether or not the behavioral experiment was performed during the mice's light or dark cycle. From the Supplementary video, linked above, the artificial lighting of the indoor facility also makes this determination impossible. As you may know, mice are nocturnal. Conducting behavioral tests during the light cycle, when the mice would normally be sleeping, can lead to dramatically different results (3,4). Interruption of a rodent’s normal sleeping period reduces welfare and increases stress (5,6). It has been recommended that for behavioral phenotyping of genetically engineered mice, dark-phase testing allows researchers to better discriminate these strains against wild-type animals and provides superior outcomes (7).

To fully assess calmness or stress, one can measure physiological parameters such as hormone levels or heart rate, to name a few. However, the authors did not examine any measure of stress beyond breathing rate, which they artificially manipulated, not even to measure the baseline stress level between groups. The use of theta rhythm as secondary external validation for emotion further supports our concerns that the authors have drawn a broad conclusion based on rather tenuous connections. The relationship between theta rhythm and arousal may depend entirely on locomotion. As noted by Biskamp and colleagues, “The power of hippocampal theta activity, which drives theta oscillations in the mPFC, depends on locomotion and is attenuated when animals remain immobile” (8). The authors conclude that mice are “calm” for simply sitting still, a behavior that has in most other cases been attributed to decreased well-being.

For the reasons listed above, we are concerned that the authors may have drawn premature and/or incorrect conclusions regarding the relative “calmness” of the mice with preBötC ablation. Importantly, the authors claim as a justification for their work that this data may be useful in understanding the effects of pranayama yoga on promoting “mental calming and contemplative states”. However, the practice of pranayama includes not only controlled breathing but also mental visualization and an increased emphasis on abdominal respiration. There are also periods when the breath is held deliberately. It cannot be assumed the various components of pranayama can individually achieve a calmer state in humans, and, crucially, these components cannot be modeled or replicated in animals.

References

1) S.D. Paolo et al., Eur J Pharmacol., 399, 43-47 (2000).

2) A.V. Kaleuff, P. Tuohimaa, Brain Res. Protoc., 13, 151-158 (2004).

3) A. Nejdi, J. M. Gustavino, R. Lalonde, Physiol. Behav., 59, 45-47 (1995).

4) A. Roedel, C. Storch, F. Holsboer, F. Ohl, Lab. Anim., 40, 371-381 (2006).

5) U. A. Abou-Ismail, O. H. P. Burman, C. J. Nicol, M. Mendl, Appl. Anim. Behav. Sci., 111, 329-341 (2008).

6) U. A. Abou-Ismail, R. A. Mohamed, S. Z, El-Kholya, Appl. Anim. Behav. Sci., 162, 47-57 (2015).

7) S. M. Hossain, B. K. Y. Wong, E. M. Simpson, Genes Brain Behav., 3, 167-177 (2004).

8) J. Biskamp, M. Bartos, J. Sauer, Sci. Rep., 7, 45508 (2017).

On 2017 Jun 05, Frances Cheng commented:

Immobility in mice, calm or stressed?

Frances Cheng, PhD; Ingrid Taylor, DVM; Emily R Trunnell, PhD

People for the Ethical Treatment of Animals

This paper by Yackle, et al. claims to have found a link between breathing and calmness in mice. However, the conclusions made by the authors leave several critical questions unanswered.

Mice are naturally inquisitive and the expression of exploratory behavior is generally interpreted as good welfare. Conversely, being motionless or less exploratory is typically thought to be indicative of stress, pain, or poor welfare. There is no established relationship between calmness and sitting still; in fact the literature would likely attribute time spent immobile to anxiety in this species. Similarly, the relationship between grooming and calmness is not clear. Grooming can be elicited by both stressful and relaxing situations, and as such is problematic to use as an absolute marker of stress levels. In some cases, grooming can be a stress reliever and restraint stress can increase grooming (1). A study by Kaleuff and Tuohima attempted to differentiate between stress grooming and relaxed grooming, stating: “While a general pattern of self-grooming uninterrupted cephalocaudal progression is normally observed in no-stress (comfort) conditions in mice and other rodents, the percentage of ‘incorrect’ transitions between different stages and the percentage of interrupted grooming bouts may be used as behavioural marker of stress" (2). Indeed the preBötC ablated mouse in this supplementary video (http://science.sciencemag.org/content/sci/suppl/2017/03/29/355.6332.1411.DC1/aai7984s1.mp4) appears to be less active, but without other objective measurements, it is a leap to conclude that this mouse is calm.

The chamber used to measure the animals’ behavior is extremely small and inadequate for observing mouse behavior and making conclusions about calmness or other emotions, particularly when the differences in behavior between the two mice being compared are very subtle, as they are here. In addition, simply being in a chamber of this size could potentially be restraining and stress-inducing. A larger chamber would allow for more traditional measures of calmness and anxiety, such as exploratory behavior, where the amount of time the mouse spends along the wall of the chamber versus the amount of time he or she leaves the safety of the walls to explore the center area is measured and scored (the Open Field test).

As mentioned, sitting still does not necessarily dictate calmness, and in many behavioral paradigms, immobility is thought to be an outward sign of anxiety or distress. The authors mention that they observe different breathing rates associated with different behaviors, e.g., faster during sniffing and slower during grooming; however, observed breathing rate alone cannot be used as the sole measure to associate an emotion with a behavior. Consider that humans under stress can hyperventilate when sitting still. It would be more informative, and more applicable to calmness, to know whether or not ablation of Cdh9/Dbx1 double-positive preBötC neurons would influence one’s ability to control their breathing and potentially to breathe slower during a psychologically stressful situation, rather than how the ablation impacts breathing coupled with normal physiological functions.

It is not clear if the experimental group is physiologically capable of breathing faster in response to external stimuli. Not being able to do so—in other words being programmed to breathe a certain way—could be distressing. The lack of compensatory respiration mechanisms, such as increased respiration in unknown, potentially dangerous situations, could affect prey species such as mice in ways that have not been previously characterized.

The experimental groups were born with full use of the Cdh9/Dbx1 double-positive preBötC neurons. These neurons were ablated in adult animals. If these animals did not have a full range of control of their breathing after ablation, they might have experienced unpleasant psychological reactions to the forced change in breathing pattern, which could be distressing.

In the Methods section, the authors did not specify whether or not the behavioral experiment was performed during the mice's light or dark cycle. From the Supplementary video, linked above, the artificial lighting of the indoor facility also makes this determination impossible. As you may know, mice are nocturnal. Conducting behavioral tests during the light cycle, when the mice would normally be sleeping, can lead to dramatically different results (3,4). Interruption of a rodent’s normal sleeping period reduces welfare and increases stress (5,6). It has been recommended that for behavioral phenotyping of genetically engineered mice, dark-phase testing allows researchers to better discriminate these strains against wild-type animals and provides superior outcomes (7).

To fully assess calmness or stress, one can measure physiological parameters such as hormone levels or heart rate, to name a few. However, the authors did not examine any measure of stress beyond breathing rate, which they artificially manipulated, not even to measure the baseline stress level between groups. The use of theta rhythm as secondary external validation for emotion further supports our concerns that the authors have drawn a broad conclusion based on rather tenuous connections. The relationship between theta rhythm and arousal may depend entirely on locomotion. As noted by Biskamp and colleagues, “The power of hippocampal theta activity, which drives theta oscillations in the mPFC, depends on locomotion and is attenuated when animals remain immobile” (8). The authors conclude that mice are “calm” for simply sitting still, a behavior that has in most other cases been attributed to decreased well-being.

For the reasons listed above, we are concerned that the authors may have drawn premature and/or incorrect conclusions regarding the relative “calmness” of the mice with preBötC ablation. Importantly, the authors claim as a justification for their work that this data may be useful in understanding the effects of pranayama yoga on promoting “mental calming and contemplative states”. However, the practice of pranayama includes not only controlled breathing but also mental visualization and an increased emphasis on abdominal respiration. There are also periods when the breath is held deliberately. It cannot be assumed the various components of pranayama can individually achieve a calmer state in humans, and, crucially, these components cannot be modeled or replicated in animals.

References

1) S.D. Paolo et al., Eur J Pharmacol., 399, 43-47 (2000).

2) A.V. Kaleuff, P. Tuohimaa, Brain Res. Protoc., 13, 151-158 (2004).

3) A. Nejdi, J. M. Gustavino, R. Lalonde, Physiol. Behav., 59, 45-47 (1995).

4) A. Roedel, C. Storch, F. Holsboer, F. Ohl, Lab. Anim., 40, 371-381 (2006).

5) U. A. Abou-Ismail, O. H. P. Burman, C. J. Nicol, M. Mendl, Appl. Anim. Behav. Sci., 111, 329-341 (2008).

6) U. A. Abou-Ismail, R. A. Mohamed, S. Z, El-Kholya, Appl. Anim. Behav. Sci., 162, 47-57 (2015).

7) S. M. Hossain, B. K. Y. Wong, E. M. Simpson, Genes Brain Behav., 3, 167-177 (2004).

8) J. Biskamp, M. Bartos, J. Sauer, Sci. Rep., 7, 45508 (2017).