Da-Jiang Zheng

Summary: Animals produce different vocalization types (e.g., contact calls, mating calls) to communicate in different behavioral contexts, but how the brain is organized to enable the production of different vocalization types remains poorly understood. Recent work found that specialized neurons in the midbrain periaqueductal gray (PAG) regulate the production of ultrasonic vocalizations (USVs) in female and male mice, but these PAG-USV neurons are not required for the production of broadband vocalizations (squeaks). To identify PAG neurons that may regulate the production of squeaks, we used an activity-dependent labeling strategy (TRAP2) to identify and manipulate PAG neurons that are active in association with the production of courtship squeaks in female mice. Optogenetic activation of courtship squeak-TRAPed PAG neurons elicits the production of squeaks, while ablation of TRAPed PAG neurons blocks the production of courtship squeaks without altering female non-vocal courtship behaviors. Surprisingly, ablation of courtship squeak-TRAPed PAG neurons did not affect the production of footshock-elicited squeaks. Moreover, non-selective PAG ablation, which blocked the production of courtship squeaks in females and USVs in both sexes, attenuated but did not block the production of footshock-elicited squeaks. Our results identify a population of PAG neurons that critically regulates the production of courtship squeaks in female mice and also suggest that a non-canonical, PAG-independent pathway regulates the production of pain-elicited vocalizations.

Animals produce different vocalization types, which differ in their acoustic features and are produced in different behavioral contexts. How vocalization-related brain circuits are organized to enable the production of different vocalization types remains poorly understood. The nucleus retroambiguus (RAm) is a hindbrain premotor region that regulates the production of both ultrasonic vocalizations (USVs) and distress calls (squeaks) in adult mice, but whether distinct or overlapping populations of RAm neurons are recruited during the production of these two vocalization types is unknown. In the current study, we used Fos immunohistochemistry to compare the counts and spatial distributions of Fos-positive RAm neurons in males and females that produced USVs and females that produced courtship squeaks. We also combined in vivo activity-dependent (TRAP2) labeling with Fos immunohistochemistry to directly compare Fos expression associated with the production of USVs and courtship squeaks in the same females. Our findings suggest that RAm contains three vocalization-related populations of neurons: squeak-related neurons, USV-related neurons, and shared neurons that are recruited during both vocalization types. These findings refine current models of the premotor control of vocalization and set the stage for future work to explore anatomical and functional heterogeneity within RAm.

Animals produce different vocalization types, which differ in their acoustic features and are produced in different behavioral contexts. How vocalization-related brain circuits are organized to enable the production of different vocalization types remains poorly understood. The nucleus retroambiguus is a hindbrain premotor region that regulates the production of both ultrasonic vocalizations (USVs) and distress calls (squeaks) in adult mice, but whether distinct or overlapping populations of RAm neurons are recruited during the production of these two vocalization types is unknown. In the current study, we used Fos immunohistochemistry to compare the counts and spatial distributions of Fos-positive RAm neurons in males and females that produced USVs and females that produced courtship squeaks. We also combined in vivo activity-dependent (TRAP2) labeling with Fos immunohistochemistry to directly compare Fos expression associated with the production of USVs and courtship squeaks in the same females. Our findings suggest that RAm contains three vocalization-related populations of neurons: squeak-related neurons, USV-related neurons, and shared neurons that are recruited during both vocalization types. These findings refine current models of the premotor control of vocalization and set the stage for future work to explore anatomical and functional heterogeneity within RAm.

Animal courtship and aggressive displays are dramatic, often sexually dimorphic behaviors that require the coordinated modulation of diverse motivational and motor circuits. In Alston’s singing mice, a novel and elaborate advertisement vocalization is sexually dimorphic and steroid sensitive ( Scotinomys teguina ). Males sing more often than females, and on average male songs have more notes. Song is influenced by circulating androgens, but how such hormonal differences influence the diverse brain regions involved in vocal display is not understood. To characterize androgen-sensitive sites in the vocal motor pathway, we used two isoforms of pseudorabies virus (PRV) to double-label circuits ending in laryngeal and jaw muscles involved in vocalization, and co-labeled these neurons for androgen receptor (AR). Next we manipulated circulating androgens and observed the effects on AR distribution and male song. We find androgens drive coordinated changes in AR abundance across motor and motivational circuits, and both individual and group differences in song are associated with AR abundance. The results reveal how circulating androgens and the auto-regulation of androgen receptors can influence the diverse circuits necessary for elaborate advertisement displays. Significance statement: Courtship and aggressive displays are among those most elaborate and dramatic of sexually dimorphic behaviors. We show that in Alston’s singing mouse, an androgen-sensitive network defined by retrograde tracing shapes vocal display effort. Our results reveal how the intensity of singing mouse vocalizations is influenced by androgen actions in the vocal motor pathway.

Vocalizations are often elaborate, rhythmically structured behaviors. Vocal motor patterns require close coordination of neural circuits governing the muscles of the larynx, jaw, and respiratory system. In the elaborate vocalization of Alston's singing mouse (Scotinomys teguina) each note of its rapid, frequency‐modulated trill is accompanied by equally rapid modulation of breath and gape. To elucidate the neural circuitry underlying this behavior, we introduced the polysynaptic retrograde neuronal tracer pseudorabies virus (PRV) into the cricothyroid and digastricus muscles, which control frequency modulation and jaw opening, respectively. Each virus singly labels ipsilateral motoneurons (nucleus ambiguus for cricothyroid, and motor trigeminal nucleus for digastricus). We find that the two isogenic viruses heavily and bilaterally colabel neurons in the gigantocellular reticular formation, a putative central pattern generator. The viruses also show strong colabeling in compartments of the midbrain including the ventrolateral periaqueductal gray and the parabrachial nucleus, two structures strongly implicated in vocalizations. In the forebrain, regions important to social cognition and energy balance both exhibit extensive colabeling. This includes the paraventricular and arcuate nuclei of the hypothalamus, the lateral hypothalamus, preoptic area, extended amygdala, central amygdala, and the bed nucleus of the stria terminalis. Finally, we find doubly labeled neurons in M1 motor cortex previously described as laryngeal, as well as in the prelimbic cortex, which indicate these cortical regions play a role in vocal production. The progress of both viruses is broadly consistent with vertebrate‐general patterns of vocal circuitry, as well as with circuit models derived from primate literature.

Vocalizations, like many social displays, are often elaborate, rhythmically structured behaviors that are modulated by a complex combination of cues. Vocal motor patterns require close coordination of neural circuits governing the muscles of the larynx, jaw, and respiratory system. In the elaborate vocalization of Alston’s singing mouse ( Scotinomys teguina ), for example, each note of its rapid, frequency-modulated trill is accompanied by equally rapid modulation of breath and gape. To elucidate the neural circuitry underlying this behavior, we introduced the polysynaptic retrograde neuronal tracer pseudorabies virus (PRV) into the cricothyroid and digastricus muscles, which control frequency modulation and jaw opening respectively. Each virus singly labels ipsilateral motoneurons (nucleus ambiguous for cricothyroid, and motor trigeminal nucleus for digastricus). We find that the two isogenic viruses heavily and bilaterally co-label neurons in the gigantocellular reticular formation, a putative central pattern generator. The viruses also show strong co-labeling in compartments of the midbrain including the ventrolateral periaqueductal grey and the parabrachial nucleus, two structures strongly implicated in vocalizations. In the forebrain, regions important to social cognition and energy balance both exhibit extensive co-labeling. This includes the paraventricular and arcuate nuclei of the hypothalamus, the lateral hypothalamus, preoptic area, extended amygdala, central amygdala, and the bed nucleus of the stria terminalis. Finally, we find doubly labeled neurons in M1 motor cortex previously described as laryngeal, as well as in the prelimbic cortex, which indicate these cortical regions play a role in vocal production. Although we observe some novel patterns of double-labelling, the progress of both viruses is broadly consistent with vertebrate-general patterns of vocal circuitry, as well as with circuit models derived from primate literature.

Because of their roles in courtship and intrasexual competition, sexual displays are often sexually dimorphic, but we know little about the mechanisms that produce such dimorphism. Among mammals, one example is the vocalization of Alston's singing mouse (Scotinomys teguina), which consists of a series of rapidly repeated, frequency-modulated notes. The rate and duration of songs is sexually dimorphic and androgen responsive. To understand the neuronal mechanisms underlying this sexual dimorphism, we map the sites of androgen sensitivity throughout the brain, focusing analysis along a pathway that spans from limbic structures to vocal motor regions. We find widespread expression of AR immunoreactivity (AR-ir) throughout limbic structures important for social behavior and vocalization, including the lateral septum, extended amygdala, preoptic area and hypothalamus. We also find extensive AR staining along previously documented vocal motor pathways, including the periaqueductal gray, parabrachial nucleus, and nucleus ambiguus, the last of which innervates intrinsic laryngeal muscles. Lastly, AR-ir is also evident in sensory areas such as the medial geniculate, inferior, and superior colliculi. A quantitative analysis revealed that males exhibited more AR-ir than females, a pattern that was most pronounced in the hypothalamus. Despite the elaboration of vocalization in singing mice, comparison with prior literature suggests that the broad pattern of AR-ir may be conserved across a wide range of rodents. Together these data identify brain nuclei well positioned to shape the sexually dimorphic vocalization of S. teguina and suggest that such androgen modulation of vocalization is evolutionary conserved among rodents.