Distribution and functional significance of laryngeal motor neurons in ultrasonic vocalizations

Abstract

Diverse vocal patterns are important to communicate in different contexts. For this, the cricothyroid (CT) and thyroarytenoid (TA) muscles are working for varying vocal frequencies and patterns. However, how the muscles are controlled by the brain and change ultrasonic vocalization (USV) frequencies are not well-studied. Adult mice emit USVs with various patterns, and this is known to be related to their behaviors, cognitive status, and disease. Therefore, we set our hypothesis that the activation of the CT and TA muscles will increase USV frequencies and mice will have distinctive distribution of motor neurons projecting to the CT or TA muscle. To check it, we investigated vocal changes by optogenetic excitation on the nucleus ambiguous (NAmb) and motor neuron localizations of the laryngeal muscles in mice. Consequently, we found that the nonspecific optogenetic excitation can lead the minimum frequency to increase and frequency range to decrease within a syllable. When tracing motor neurons of the laryngeal muscles with cholera toxin subunit-b and pseudorabies virus, we observed that motor neurons of each muscle were distributed differently. CT motor neurons were clustered compactly in the rostral NAmb while TA motor neurons were scattered widely in the NAmb. Furthermore, some motor neurons that innervate the TA or CT muscle were co-localized in the rostral NAmb. This study supports our hypothesis on the involvement of motor neurons of the CT and TA muscles in the USV pattern modulation and unique localization of the motor neurons in the NAmb. The results also provide fundamental and essential information for understanding the mechanisms of mouse USVs. The findings will be further validated with additional observations such as optogenetic experiments targeting motor neurons of a specific muscle, more specific injection of tracers or viruses, and acquisition of laryngeal histology to check the injection accuracy.