Optogenetic stimulation of postinspiratory complex (PiCo) reveals hub for postinspiratory laryngeal closure and swallow related behaviors

Abstract

Little is known about the central control of postinspiration, a phase critical for vocalization, cough, swallow and laryngeal control (closure). Laryngeal activity serves dual purposes for airway protection during swallow and for mechanical slowing of expiration. Dysregulation of laryngeal activity during postinspiratory behaviors can allow foreign material/food/liquid to enter the larynx and trachea (aspiration) and cause pneumonia, a leading cause of death among many central nervous system disorders. Schluckatmung (swallow breath) is the activation of the diaphragm during swallowing which creates a negative trans-diaphragmatic pressure aiding in the transition of the bolus from the pharynx to the esophagus. The Postinspiratory complex (PiCo), a medullary region composed of neurons that uniquely co-express both cholinergic and glutamatergic markers, has been shown to be active during postinspiration, reset the respiratory rhythm when stimulated, and is proposed as the pattern generator for postinspiration. However, the role of PiCo in other postinspiratory behaviors such as swallow and the neural coordination of inspiration, postinspiration and swallow is poorly understood. Our preliminary data revealed that activation of PiCo can trigger not only postinspiration, but also swallow, and both behaviors reset the respiratory rhythm. To evaluate the role PiCo plays in postinspiratory behaviors we targeted both cholinergic and glutamatergic neurons by optogenetically stimulating PiCo in anesthetized spontaneously breathing in-vivo adult ChATcre/Ai32(ChR2) and Vglut2cre/Ai32 mice. The ventral surface of the brainstem was exposed to place bilateral glass fiber optics connected to a blue (447nm) laser over the ventral surface in the region of PiCo using pulse durations ranging from 40-200ms. Swallow and postinspiratory laryngeal activity were measured via monopolar suction electrodes of the hypoglossal (XII) and vagus (X) nerves as well as bipolar electromyogram (EMG) of the submental and laryngeal complex and costal diaphragm muscles. We found that optogenetic stimulation of ChAT neurons applied at random across different phases of breathing resulted in a significantly higher probability of triggering a swallow during inspiration than triggering laryngeal closure (p=0.02). Reset of the respiratory rhythm is contingent upon stimulation of either swallow or laryngeal closure, whereas no behavioral response to the laser pulse results in an unchanged respiratory rhythm. We found that stimulating Vglut2 neurons in PiCo evoked both swallow and laryngeal postinspiratory activity. Of the 97 swallows triggered by optogenetic stimulation of Vglut2 neurons, schluckatmung was present in 67 swallows, whereas schluckatmung is never triggered during ChAT stimulation at PiCo. Our data indicates that PiCo is a hub for the generation of both postinspiratory activity and swallowing. The activation of swallowing may involve neurons in the nTS implicated in the generation of swallowing.