The breath shape controls intonation of mouse vocalizations

Reviewer #1 (Public Review):

Summary:
In this important work, the authors propose and test a model for the control of murine ultrasonic vocalizations (USV) in which two independent mechanisms, involving changes in laryngeal opening or airflow, control vocal tone. They present compelling experimental evidence for this dual control model by demonstrating the ability of freely behaving adult mice to generate vocalizations with various intonations by modulating both the breathing pattern and the laryngeal muscles. They also present novel evidence that these mechanisms are encoded in the brainstem vocalization central neural pattern generator, particularly in the component in the medulla called the intermediate reticular oscillator (iRO). The results presented clearly advance understanding of the developmental nature of the iRO, its ability to intrinsically generate and control many of the dynamic features of USV including those related to intonation, and its coordination with/control of expiratory airflow patterns. This work will interest neuroscientists investigating the neural generation and control of vocalization, breathing, and more generally, neuromotor control mechanisms.

Strengths:
Important features and novelty of this work include:

1. The study employs an effective combination of anatomical, molecular, and functional/ behavioral approaches to examine the hypothesis and provide novel data indicating that variations in expiratory airflow can change the pitch patterns of adult murine USV.

2. The results significantly extend the authors' previous work that identified the iRO in neonatal mice by now presenting data that functionally demonstrates the existence of the critical Penk+Vglut2+ iRO neurons in adult mice, indicating that the iRO neurons maintain their function in generating vocalization throughout development.

3. The results convincingly demonstrate that the iRO neurons encode and can generate vocalizations by modulating both breathing and the laryngeal muscles.

4. The anatomical mapping and tracing results establish an important set of input and output circuit connections to the iRO, including input from the vocalization-promoting subregions of the midbrain periaqueductal gray (PAG), as well as output axonal projections to laryngeal motoneurons, and to the respiratory rhythm generator in the preBötzinger complex.

5. These studies advance the important concept that the brainstem vocalization pattern generator integrates with the medullary respiratory pattern generator to control expiratory airflow as a key mechanism to produce various USV types characterized by different pitch patterns.

Weaknesses:
A limitation is that the cellular and circuit mechanisms by which the vocalization pattern generator integrates with the respiratory pattern generator to control expiratory airflow have not been fully worked out, requiring future studies.

Reviewer #2 (Public Review):

Summary:
Both human and non-human animals modulate the frequency of their vocalizations to communicate important information about context and internal state. While regulation of the size of the laryngeal opening is a well-established mechanism to regulate vocal pitch, the contribution of expiratory airflow to vocal pitch is less clear. To consider this question, this study first characterizes the relationship between the dominant frequency contours of adult mouse ultrasonic vocalizations (USVs) and expiratory airflow using whole-body plethysmography. Next, the authors build off of their previous work characterizing intermediate reticular oscillator (iRO) neurons in mouse pups to establish the existence of a genetically similar population of neurons in adults and show that artificial activation of iRO neurons elicits USV production in adults. Third, the authors examine the acoustic features of USV elicited by optogenetic activation of iRO and find that a majority of natural USV types (as defined by pitch contour) are elicited by iRO activation.

Strengths:
Strengths of the study include the novel consideration of expiratory airflow as a mechanism to regulate vocal pitch and the use of intersectional methods to identify and activate the iRO in adult mice. The establishment of iRO neurons as a brainstem population that regulates vocal production across development is an important finding.

Weaknesses:
The study does not include statistical analyses to compare the observed relationships between expiratory airflow and USV pitch to a null model in which expiratory airflow and USV pitch are unrelated. The findings of the study also do not provide clear evidence to support the authors' model in which distinct brainstem populations (iRO and RAm) independently regulate expiratory airflow and laryngeal adduction. Although this study establishes iRO as an important population that regulates USV production in adult mice, the question of whether and how different brainstem populations contribute differentially to vocal production remains an important open question. Lastly, the addition of statistical analyses would help to strengthen the study's conclusion that iRO activation positively biases the relationship between expiratory airflow and USV pitch across multiple USV types.