Selective transduction and photoinhibition of pre-Bötzinger complex neurons that project to the facial nucleus in rats affects nasofacial activity
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eLife assessment
This important study advances our understanding of the composition and circuit organization of the preBötzinger complex (preBötC)-the brainstem region that generates the respiratory rhythm and coordinates breathing with different motor and physiological behaviors in mammals. The reviewers agreed that the evidence supporting the conclusion that the preBötC is composed of a segregated subgroup of output neurons that modulates orofacial muscle activity is compelling and based on technically elegant, state-of-the-art combinatorial dual viral transgenic and optogenetic approaches in rats. After the cytoarchitectonic analyses are strengthened, the work will be of interest to neuroscientists and physiologists working on the neural control of breathing and other motor systems.
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Abstract
The pre-Bötzinger complex (preBötC), a key primary generator of the inspiratory breathing rhythm, contains neurons that project directly to facial nucleus (7n) motoneurons to coordinate orofacial and nasofacial activity. To further understand the identity of 7n-projecting preBötC neurons, we used a combination of optogenetic viral transgenic approaches to demonstrate that selective photoinhibition of these neurons affects mystacial pad activity, with minimal effects on breathing. These effects are altered by the type of anesthetic employed and also between anesthetized and conscious states. The population of 7n-projecting preBötC neurons we transduced consisted of both excitatory and inhibitory neurons that also send collaterals to multiple brainstem nuclei involved with the regulation of autonomic activity. We show that modulation of subgroups of preBötC neurons, based on their axonal projections, is a useful strategy to improve our understanding of the mechanisms that coordinate and integrate breathing with different motor and physiological behaviors. This is of fundamental importance, given that abnormal respiratory modulation of autonomic activity and orofacial behaviors have been associated with the development and progression of diseases.
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eLife assessment
This important study advances our understanding of the composition and circuit organization of the preBötzinger complex (preBötC)-the brainstem region that generates the respiratory rhythm and coordinates breathing with different motor and physiological behaviors in mammals. The reviewers agreed that the evidence supporting the conclusion that the preBötC is composed of a segregated subgroup of output neurons that modulates orofacial muscle activity is compelling and based on technically elegant, state-of-the-art combinatorial dual viral transgenic and optogenetic approaches in rats. After the cytoarchitectonic analyses are strengthened, the work will be of interest to neuroscientists and physiologists working on the neural control of breathing and other motor systems.
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Reviewer #1 (Public Review):
This paper by Melo et al. is a technically elegant study investigating the important emerging hypothesis that the brainstem preBötzinger complex (preBötC) region - a critical nuclear structure where the rhythm of breathing in mammals originates - has segregated subgroups of output neurons that modulate specific behaviors coordinated with breathing, in this study the orofacial muscle activity. The preBötC has been under intense investigation for several decades but the subregional neuronal subtype composition and organization are not fully understood. Understanding this organization and how breathing modulates specific behaviors has many implications for normal brain function and pathophysiology.
Strengths of the paper include:
- The authors use an effective combinatorial dual viral transgenic approach for …
Reviewer #1 (Public Review):
This paper by Melo et al. is a technically elegant study investigating the important emerging hypothesis that the brainstem preBötzinger complex (preBötC) region - a critical nuclear structure where the rhythm of breathing in mammals originates - has segregated subgroups of output neurons that modulate specific behaviors coordinated with breathing, in this study the orofacial muscle activity. The preBötC has been under intense investigation for several decades but the subregional neuronal subtype composition and organization are not fully understood. Understanding this organization and how breathing modulates specific behaviors has many implications for normal brain function and pathophysiology.
Strengths of the paper include:
- The authors use an effective combinatorial dual viral transgenic approach for Cre-dependent expression of the chloride channel (GtACR2) and labeling of neurons projecting to the facial motor nucleus controlling orofacial muscle activity, for optogenetic photoinhibition of these preBötC neurons in vivo.
- The experimental results presented convincingly support the authors' conclusion that a subgroup of preBötC neurons provides inspiratory modulation of facial motoneurons that appear to be distinct from other output neurons that drive inspiratory activity to bulbospinal neurons and neurons projecting to autonomic nervous system circuits.
- These results advance our understanding of preBötC circuit organization that coordinates and integrates breathing with different motor and physiological behaviors.
Weaknesses:
There are a few technical issues related to the photoinhibition paradigm used and the patterns of neuronal transduction with the dual viral transgenic approach used that the authors need to clarify. -
Reviewer #2 (Public Review):
By using elegant optogenetic viral transgenic approaches the authors show that subgroups of neurons located in the preBötzinger region of the brainstem and projecting to the facial nucleus are involved in controlling orofacial activity while being minimally implicated in breathing behavior. The experiments are properly performed, and technically challenging with several physiological parameters measured in vivo allowing the monitoring of several functions simultaneously (breathing, heart rate, blood pressure, orofacial muscle activity). They also demonstrate that the type of anesthetic used and the state of consciousness are important for the effects of their photoinhibition. While this study is particularly interesting for a better understanding of the coordination between breathing and other behaviours …
Reviewer #2 (Public Review):
By using elegant optogenetic viral transgenic approaches the authors show that subgroups of neurons located in the preBötzinger region of the brainstem and projecting to the facial nucleus are involved in controlling orofacial activity while being minimally implicated in breathing behavior. The experiments are properly performed, and technically challenging with several physiological parameters measured in vivo allowing the monitoring of several functions simultaneously (breathing, heart rate, blood pressure, orofacial muscle activity). They also demonstrate that the type of anesthetic used and the state of consciousness are important for the effects of their photoinhibition. While this study is particularly interesting for a better understanding of the coordination between breathing and other behaviours controlled by neurons located in the brainstem, the identification of the neurons of interest here as components of the preBötC network requests clarification and the interpretation of the effects of photo-inhibiting both excitatory and inhibitory neurons remain difficult.
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Reviewer #3 (Public Review):
Melo et. al. sought to characterize the neuronal basis for the breathing modulation of nasal dilation (mystacial pad activity). The hypothesis is that a subset of breathing pacemaker neurons (preBötC) are specialized to relay a breathing signal to modulate the nares instead of contributing to pacing breathing. The authors identify that a subset of neurons within the anatomical region of the preBötC project to the facial motor nucleus and are required for the respiratory modulation of the nares. Furthermore, they show these neurons are partially required for breathing. The authors do this by using an intersectional genetic approach to selectively inhibit the preBötC neurons that project to the facial motor nucleus while measuring the impact of this manipulation on the breathing-related movement of the nares …
Reviewer #3 (Public Review):
Melo et. al. sought to characterize the neuronal basis for the breathing modulation of nasal dilation (mystacial pad activity). The hypothesis is that a subset of breathing pacemaker neurons (preBötC) are specialized to relay a breathing signal to modulate the nares instead of contributing to pacing breathing. The authors identify that a subset of neurons within the anatomical region of the preBötC project to the facial motor nucleus and are required for the respiratory modulation of the nares. Furthermore, they show these neurons are partially required for breathing. The authors do this by using an intersectional genetic approach to selectively inhibit the preBötC neurons that project to the facial motor nucleus while measuring the impact of this manipulation on the breathing-related movement of the nares and breathing. As a control, the authors broadly silence the preBötC. The simplicity of the experiments makes the results robust and the correct positive control is used. The manuscript's conclusion contributes to the logic for the breathing modulation of the nares and the notion that subsets of neurons in the preBötC play distinct roles in breathing-related behaviors. Although the data are compelling for this conclusion, alternative models cannot be completely ruled out, like that these neurons are important for breathing rhythm generation and a secondary cell type from other premotor centers (Kurnikova 2019) are those that relay this signal to the motor neurons for the nares. The role of the preBötC as a "master clock" for orofacial activity (nose movement, swallowing, chewing, vocalizing; Kurnikova 2017) is an important line of research and this work contributes to understanding the cellular mechanisms.
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