Sigh generation in preBötzinger Complex

Reviewer #1 (Public Review):

This manuscript validates and extends upon the sigh-generating circuit between the NMB/GRP+ RTN/parafacial neurons and the NMBR/GRPR+ preBötC neurons established in Li et al., 2016. The authors generate multiple transgenic lines that enable selective targeting of these various sub-populations of cells and demonstrate the sufficiency of each type in generating a sigh breath. Additionally, they show that NMBR and GPRP preBötC neurons are glutamatergic, have overlapping and distinct expressions, and do not express SST. Beyond this validation, the authors show that ectopic stimulation of SST neurons is sufficient to evoke sighs and that they are necessary for NMB/GRP-induced sighing. This data is the first time that preBötC neurons downstream of NMBR/GRPR neurons have been identified.

The five conclusions stated at the end of the introduction are supported by the data, but a strong emphasis throughout the manuscript is the identification of an unsubstantiated slow sigh rhythm that is produced by NMBR/GRPR neurons. To make such a novel (and quite surprising) claim requires many more studies and the conclusion is dependent on how the authors have defined a sigh. Moreover, some data within the paper conflicts with this idea.

In summary, the optogenetic and chemogenetic characterization of the neuropeptide pathway transgenic lines nicely aligns with and provides important validation of the previous study by Li et. al., 2016 and the SST neuron studies provide a new mechanism for the transformation of NMBR/GRPR neuropeptide activation into a sigh. These are important findings and they should be the points emphasized. The proposal of a slow sigh rhythm should be more rigorously established with new experiments and analysis or should be more carefully described and discussed.

Reviewer #2 (Public Review):

Summary:

This study investigates in mice neural mechanisms generating sighs, which are periodic large-amplitude breaths occurring during normal breathing that subserve physiological pulmonary functions and are associated with emotional states such as relief, stress, and anxiety. Sighs are generated by a structure called the preBötzinger complex (preBötC) in the medulla oblongata that generates various forms of inspiratory activity including sighs. The authors have previously described a circuit involving neurons producing bombesin-related peptides Neuromedin B (NMB) and gastrin-releasing peptide (GRP) that project to preBötC neurons expressing receptors for NMB (NMBRs) and GRP (GRPRs) and that activation of these preBötC neurons via these peptide receptors generates sighs. In this study, the authors further investigated mechanisms of sigh generation by applying optogenetic and chemogenetic strategies to selectively activate the subpopulations of preBötC neurons expressing NMBRs and/or GRPRs, and a separate subpopulation of neurons expressing somatostatin (SST) but not NMBRs and GRPRs. The authors present convincing evidence that sigh-like inspirations can be evoked by photostimulation of the preBötC neurons expressing NMBRs or GRPRs. Photostimulation of SST neurons can independently evoke sighs, and chemogenetic inhibition of these neurons can abolish sighs. The results presented support the authors' conclusion that the preBötC neurons expressing NMBRs or GRPRs produce sighs via pathways to downstream SST neurons. Thus, these studies have identified some of the preBötC cellular elements likely involved in generating sighs.

Strengths:

(1) This study employs an effective combination of electrophysiological, transgenic, optogenetic, chemogenetic, pharmacological, and neuron activity imaging techniques to investigate sigh generation by distinct subpopulations of preBötC neurons in mice.

(2) The authors extend previous studies indicating that there is a peptidergic circuit consisting of NMB and GRP expressing neurons that project from the parafacial (pF) nucleus region to the preBötC and provides sufficient input to generate sighs, since photoactivation of either pF NMB or GRP neurons evoke ectopic sighs in this study.

(3) Convincing evidence is presented that sighs can be evoked by direct photostimulation of preBötC neurons expressing NMBRs and/or GRPRs, and also a separate subpopulation of neurons expressing somatostatin (SST) but not NMBRs and GRPRs.

(4) The mRNA-expression data presented from in situ hybridization indicates that most preBötC neurons expressing NMBR, GRPR (or both) are glutamatergic and excitatory.

(5) Measurements in slices in vitro indicate that only the NMBR-expressing neurons are normally rhythmically active during normal inspiratory activity and endogenous sigh activity.

(6) Evidence is presented that activation of preBötC NMBRs and/or GRPRs is not necessary for sigh production, suggesting that sighs are not the unique product of the preBötC bombesin-peptide signaling pathway.

(7) The novel conclusion is presented that the preBötC neurons expressing NMBRs and/or GRPRs produce sighs via the separate downstream population of preBötC SST neurons, which the authors demonstrate can independently generate sighs, whereas chemogenetic inhibition of preBötC SST neurons selectively abolishes sighs generated by activating NMBRs and GRPRs.

Weaknesses:

(1) While these studies have identified subpopulations of preBötC neurons capable of episodically evoking sigh-like inspiratory activity, mechanisms producing the normal slow sigh rhythm were not investigated and remain unknown.

(2) Several key technical aspects of the study require further clarification to aid in interpreting the experimental results, including issues relating to the validation of the transgenic mouse lines and virally transduced expressions of proteins utilized for optogenetic and chemogenetic experiments, as well as justifying the optogenetic photostimulation paradigms used to evoke sighs.

Reviewer #3 (Public Review):

Summary:

This manuscript by Cui et al., studies the mechanisms for the generation of sighing, an essential breathing pattern. This is an important and interesting topic, as sighing maintains normal pulmonary function and is associated with various emotional conditions. However, the mechanisms of its generation remain not fully understood. The authors employed different approaches, including optogenetics, chemogenetics, intersectional genetic approach, slice electrophysiology, and calcium imaging, to address the question, and found several neuronal populations are sufficient to induce sighing when activated. Furthermore, ectopic sighs can be triggered without the involvement of neuromedin B (NMB) or gastrin-releasing peptide (GRP) or their receptors in the preBötzinger Complex (preBötC) region of the brainstem. Additionally, activating SST neurons in the preBötC region induces sighing, even when other receptors are blocked. Based on these results, the authors concluded that increased excitability in certain neurons (NMBR or GRPR neurons) activates pathways leading to sigh generation, with SST neurons serving as a downstream component in converting regular breaths into sighs

Strengths:

The authors employed a combination of various sophisticated approaches, including optogenetics, chemogenetics, intersectional genetic approach, slice electrophysiology and calcium imaging, to precisely pinpoint the mechanism responsible for sigh generation. They utilized multiple genetically modified mouse lines, enabling them to selectively manipulate and observe specific neuronal populations involved in sighing.

Using genetics and calcium imaging, the authors record the neuronal activity of NMBR and GRPR neurons, respectively, and identify their differences in activity patterns. Furthermore, by applying the intersectional approach, the authors were able to genetically target and manipulate several distinct neuronal populations, such as NMBR+, GRPR- neurons, and GRPR+, NMBR- neurons, and conducted a detailed characterization of their functions in influencing sighing.

Weaknesses:

The authors combined multiple approaches in this manuscript; however, the rationale and experimental details require further explanation, and their impacts on the conclusion require clarification. For instance, how and why the variability in optogenetic activation conditions could impact the experimental outcomes. Additionally, a more detailed characterization of the viral labeling efficiency and specificity is necessary to validate the claims made in these experiments. Without this, the results could be compromised by potential discrepancies in the number of labeled neurons or unintended labeling of other populations.

Moreover, the conclusion that preBötC NMBR and GRPR activations are unnecessary for sighing is not fully supported by the current experimental design. While the study shows that sighing can still be induced despite pharmacological inhibition of NMBR and GRPR, this does not conclusively prove that these receptors are not required under natural conditions. The artificial activation of downstream pathways through optogenetic or chemogenetic methods does not negate the potential physiological role of these receptors in sigh production. Therefore, the interpretation of these findings should be approached with caution, and further investigation is warranted to definitively determine the necessity of NMBR and GRPR activations in the natural sighing process.