Genetic identification of a hindbrain nucleus essential for innate vocalization

Abstract

Vocalization in young mice is an innate response to isolation or mechanical stimulation. Neuronal circuits that control vocalization and breathing overlap and rely on motor neurons that innervate laryngeal and expiratory muscles, but the brain center that coordinates these motor neurons has not been identified. Here, we show that the hindbrain nucleus tractus solitarius (NTS) is essential for vocalization in mice. By generating genetically modified newborn mice that specifically lack excitatory NTS neurons, we show that they are both mute and unable to produce the expiratory drive required for vocalization. Furthermore, the muteness of these newborns results in maternal neglect. We also show that neurons of the NTS directly connect to and entrain the activity of spinal (L1) and nucleus ambiguus motor pools located at positions where expiratory and laryngeal motor neurons reside. These motor neurons control expiratory pressure and laryngeal tension, respectively, thereby establishing the essential biomechanical parameters used for vocalization. In summary, our work demonstrates that the NTS is an obligatory component of the neuronal circuitry that transforms breaths into calls.V ocalization is the primary mechanism used by many vertebrate species for communication (1). Whereas adult mice call during courtship, mating, and territorial disputes, newborn mice use vocalization to communicate with their mothers (2, 3). Newborn mice, when isolated, produce ultrasonic calls (USCs) that elicit search and retrieval behavior by their mothers. Thus, vocalizations of newborn mice represent an innate behavior that is thought to rely on a genetically determined circuit. Such innate vocalizations are reminiscent of nonverbal utterances of humans like laughing, crying, sighing, and moaning.The central circuits that control vocalization have been widely studied in adult vertebrates, where they overlap in their executive components with respiratory circuits (4). Forebrain pathways that control the frequency and sequence of ultrasounds in mice are not essential for innate vocalization (5, 6); rather, it is the periaqueductal gray in the midbrain that modulates the activity of motor neurons in the hindbrain and spinal cord to implement calls and modulate breathing (7,8). Calls are shaped through a biomechanical process that involves variations in subglottal air pressure and laryngeal muscle tension (9, 10). Expiration is an important determinant of subglottal air pressure (11), suggesting that expiratory muscle activity and laryngeal tension are highly coordinated during vocalization. However, because expiratory and laryngeal motor neurons are located at markedly different axial levels of the nervous system, in the spinal cord (T11-L1 levels, expiratory) and hindbrain (nucleus ambiguus, laryngeal), how the activities of these motor pools are coordinated is unclear (12, 13). More importantly, the identity and location of functionally important premotor neurons for vocalization are little known.Using mouse genetics to investigate the ...

The postnatal development of ultrasonic vocalization‐associated breathing is altered in glycine transporter 2‐deficient mice

“…By contrast, USV in neonatal mice requires recruitment of abdominal muscles (Hernandez‐Miranda et al . ) and thus it occurs, per definition (Bianchi et al . ) during active expiration.…”

“…These are characterized by an increase of inspiratory and particularly expiratory air flow (see Methods). Such intermittent alterations of breathing have been shown to be related to USV (Hodges et al 2009;Hernandez-Miranda et al 2017). We refer to these alterations of breathing as NVL cycles.…”

“…At birth, USV-calls are associated with active expiration (Hernandez-Miranda et al 2017) and thus with an increase of airway pressure and expiratory flow. Moreover, it is suggested that the peak frequency of USVs depends on the speed of the expiratory airflow (Mahrt et al 2016).…”

“…However, some investigators report that generation of post-inspiratory activity depends on GABAergic and not glycinergic inhibition in mice (Anderson et al 2016). By contrast, USV in neonatal mice requires recruitment of abdominal muscles (Hernandez-Miranda et al 2017) and thus it occurs, per definition (Bianchi et al 1995) during active expiration. Active expiration is absent in quiet breathing but is generated, apart from vocalization, during coughing and sneezing, as well as during states of high metabolic demand (Price & Batsel, 1970;Richter, 1982;Anderson & Ramirez, 2017).…”

Monosynaptic projections to excitatory and inhibitory preBötzinger Complex neurons

“…The NTS receives sensory signals from vagus, facial, hypoglossal and glossopharyngeal nerves, as well as peripheral chemo-, baro-, and mechanoreceptors; it also receives descending input from the forebrain originating from hypothalamus, including the paraventricular nucleus, amygdala (basolateral, central), and cortex (Affleck, Coote, & Pyner, 2012;Schwaber, Kapp, Higgins, & Rapp, 1982;van der Kooy, Koda, McGinty, Gerfen, & Bloom, 1984) that can modulate the processing of these sensory signals. The NTS also contains premotoneurons critical for coordinating expiratory breaths for vocalizations (Hernandez-Miranda et al, 2017). From the pons, the preBötC receives direct input from parabrachial and KF nuclei, which play a role in regulating the timing and phase transitions of the respiratory cycle.…”