Genetic identification of a hindbrain nucleus essential for innate vocalization Luis Rodrigo Hernandez-Mirandaa,1,2, Pierre-Louis Ruffaulta,1, Julien C. Bouvierb, Andrew J. Murrayc, Marie-Pierre Morin-Surunb, Niccolò Zampieria, Justyna B. Cholewa-Waclawa, Elodie Eyd, Jean-Francois Brunete, Jean Champagnatb,2, Gilles Fortinb,2, and Carmen Birchmeiera,2 aMax Delbrueck Center for Molecular Medicine in the Helmholtz Association, 13125 Berlin, Germany; bParis-Saclay Institute for Neuroscience, UMR9197/ CNRS, 91190 Gif sur Yvette, France; cHoward Hughes Medical Institute, Columbia University, New York, NY 10032; dPasteur Institute, 75015 Paris, France; and eInstitut de Biologie de l’École Normale Supérieure, 75005 Paris, France Edited by Joshua R. Sanes, Harvard University, Cambridge, MA, and approved June 16, 2017 (received for review February 22, 2017) Vocalization in young mice is an innate response to isolation or Using mouse genetics to investigate the neuronal basis of in- mechanical stimulation. Neuronal circuits that control vocalization nate vocalization, we identified the nucleus tractus solitarius and breathing overlap and rely on motor neurons that innervate (NTS) as a crucial vocal nucleus. Newborn mice that lack the laryngeal and expiratory muscles, but the brain center that NTS are mute (i.e., unable to call), and do not receive ade- coordinates these motor neurons has not been identified. Here, we quate maternal care; however, these mice do display other show that the hindbrain nucleus tractus solitarius (NTS) is essential for vocalization-associated behaviors, such as mouth opening and vocalization in mice. By generating genetically modified newborn the production of clicks. We found that in these mice, mute- mice that specifically lack excitatory NTS neurons, we show that they ness is accompanied by a deficit in the ability to generate the are both mute and unable to produce the expiratory drive required expiratory pressure necessary for vocalization. We also show for vocalization. Furthermore, the muteness of these newborns that the NTS directly connects to and activates spinal (L1) and results in maternal neglect. We also show that neurons of the NTS nucleus ambiguus motor neurons at positions where expira- directly connect to and entrain the activity of spinal (L1) and nucleus tory and laryngeal motor neurons reside (12, 13). Our data ambiguus motor pools located at positions where expiratory and laryngeal motor neurons reside. These motor neurons control suggest that NTS neurons establish the biomechanical pa- NEUROSCIENCE expiratory pressure and laryngeal tension, respectively, thereby rameters essential for call production by controlling the ac- establishing the essential biomechanical parameters used for tivity of expiratory and laryngeal motor neurons. Thus, we vocalization. In summary, our work demonstrates that the NTS is identify the NTS as an obligatory component of the circuit an obligatory component of the neuronal circuitry that transforms that links breathing and vocalization. breaths into calls. Results vocalization | expiration | hindbrain | premotor neurons | Olig3 Identification of Hindbrain Neurons Associated with Vocalization. The vocalizations of young mice in the postnatal period pro- ocalization is the primary mechanism used by many verte- vides a model for exploring the neuronal circuitry responsible Vbrate species for communication (1). Whereas adult mice for this behavior. When isolated, newborn mice produce USCs call during courtship, mating, and territorial disputes, newborn mice use vocalization to communicate with their mothers (2, 3). Significance Newborn mice, when isolated, produce ultrasonic calls (USCs) that elicit search and retrieval behavior by their mothers. Thus, Vocalization is a primary method of communication in many vocalizations of newborn mice represent an innate behavior that species and relies on coordinated muscle activity. Vocalization is thought to rely on a genetically determined circuit. Such innate and breathing must be synchronized, because calls can be vocalizations are reminiscent of nonverbal utterances of humans evoked only during expiration. How vocalization and breath- like laughing, crying, sighing, and moaning. ing are coordinated is not well understood. Here, we show that The central circuits that control vocalization have been widely newborn mice with impaired development of the nucleus tractus solitarius (NTS) are mute and cannot generate the ex- studied in adult vertebrates, where they overlap in their executive piratory pressure needed for vocalization. Furthermore, they components with respiratory circuits (4). Forebrain pathways do not receive appropriate maternal care. We demonstrate that that control the frequency and sequence of ultrasounds in mice the NTS contains premotor neurons that directly project to and are not essential for innate vocalization (5, 6); rather, it is the entrain the activity of spinal (L1) and nucleus ambiguus motor periaqueductal gray in the midbrain that modulates the activity neurons known to control expiratory pressure and laryngeal of motor neurons in the hindbrain and spinal cord to implement tension, respectively. We conclude that the NTS is an essential calls and modulate breathing (7, 8). Calls are shaped through a component of the vocal circuit. biomechanical process that involves variations in subglottal air pressure and laryngeal muscle tension (9, 10). Expiration is an Author contributions: L.R.H.-M., J.C., G.F., and C.B. designed research; L.R.H.-M., P.-L.R., important determinant of subglottal air pressure (11), suggesting A.J.M., and J.C. performed research; J.C.B., A.J.M., M.-P.M.-S., N.Z., J.B.C.-W., E.E., and J.-F.B. contributed new reagents/analytic tools; L.R.H.-M., J.C., and C.B. analyzed data; that expiratory muscle activity and laryngeal tension are highly and L.R.H.-M., G.F., and C.B. wrote the paper. coordinated during vocalization. However, because expiratory The authors declare no conflict of interest. and laryngeal motor neurons are located at markedly different This article is a PNAS Direct Submission. axial levels of the nervous system, in the spinal cord (T11– Freely available online through the PNAS open access option. L1 levels, expiratory) and hindbrain (nucleus ambiguus, laryn- 1L.R.H.-M. and P.-L.R. contributed equally to this work. geal), how the activities of these motor pools are coordinated is 2To whom correspondence may be addressed. Email: [email protected]; Jean. unclear (12, 13). More importantly, the identity and location of [email protected]; [email protected]; or [email protected]. functionally important premotor neurons for vocalization are This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. little known. 1073/pnas.1702893114/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1702893114 PNAS Early Edition | 1of6 Downloaded by guest on September 26, 2021 and short clicks (Fig. 1A; spectrograms illustrate sound frequen- cies and intensity of calls). Calls are particularly frequent during the first hours of life (Fig. S1). Newborn mice also vocalize with a combination of stereotypic broadband audible calls (ACs), USCs, and short clicks in response to mechanical stimulation (Fig. 1A). Given the central role of hindbrain circuits in breathing and breathing-associated behaviors, we analyzed call production in mutant mouse strains that display deficits in hindbrain develop- ment. In doing so, we identified two strains, Oligodendrocyte transcription factor 3 (Olig3) and T-cell leukemia homeobox 3 (Tlx3) mutants, with severe vocalization impairment (Fig. 1A) (14, 15). Whereas Tlx3 mutants produced weak and rare calls, Olig3 mutant newborns produced no calls either after mechanical stimulation or when isolated from the mother (Fig. 1 B and C). Both mutants were able to generate other behaviors associated with vocalization, including mouth openings and click production (Fig. 1B). This indicates that these mice have a deficit in the neu- ronal circuitry that specifically controls the production of calls. Previous studies have shown that mutations in Olig3 and Tlx3 affect the development of largely nonoverlapping neuronal pop- ulations in the mouse hindbrain (14, 15). The single exception is the dA3 neuronal type produced exclusively in rhombomeres 4–7, which is impaired in both mutant strains. This raises the pos- sibility that dA3 neurons are required for call production. The Olig3 transcription factor is expressed in a dorsal hindbrain – Fig. 2. NTS neurons are essential for vocalization. (A, Left) Transverse section progenitor domain that generates dA1 dA4 neuronal types; of rhombomere 7 stained with Olig3 antibodies (green) and DAPI (blue). among these, dA3 neurons express the Tlx3 transcription factor Olig3 is expressed in dorsal progenitor cells at E10.5. (A, Right) Scheme showing (Fig. 2A). dA3 neurons contribute to four distinct hindbrain cen- genes expressed in progenitor cells and neurons of the dorsal hindbrain. Olig3+ ters: the NTS, area postrema, and A1/C1 and A2/C2 adrenergic progenitors generate dA1-4 neurons; dA3 neurons express Tlx3 and Phox2b. groups (shown schematically in Fig. 2B) (14–17). During devel- (B) Scheme of a transverse hindbrain section depicting dA3 neuronal derivatives + opment, all dA3 neurons are excitatory (vGluT2 ) and coexpress (green): area postrema (AP), NTS, A1/C1, and A2/C2 adrenergic groups. the Paired-like homeobox 2b (Phox2b) (Fig. S2 A–K). Thus, all (C) Column plot