Mechanisms Underlying Phrenic Pattern Formation
Authors:   , Ghali Michael George Zaki , Houle John D , Marchenko Vitaliy 
Published: January 2022

The diaphragm is the primary respiratory muscle underlying alveolar ventilation in mammals. Its contraction is controlled by the phrenic motor system. Normal breathing, also referred to as eupnea, is characteristically triphasic, consisting of inspiratory, post-inspiratory, and late-expiratory epochs. The phrenic nerve discharges augmentatively during inspiration, occasionally exhibits decrementing post-inspiratory activity, and is mostly silent during late-expiration. While brainstem medullary respiratory nuclei certainly generate this rhythm and pattern, intraspinal mechanisms contributing to shaping of the final phrenic pattern remain poorly understood, as much emphasis has historically centered on supraspinal control. In the first part of this work, we seek to synthesize the vast literature concerning organization of the phrenic motor system to provide an integrated and comprehensive framework in which the functional implications deriving from the findings of our studies may be broadly contextualized and understood. In the second part of this work, we seek to investigate mechanisms underlying eupneic phrenic pattern formation in bulbospinal-intact and hemisected animals. Lastly, we seek to test the hypothesis that spinal mechanisms are capable of generating phrenic motor activity absent supraspinal drive in the adult rat, characterizing induced and spontaneous patterns of phrenic nerve discharge in high-cervically transected animals. The phrenic nucleus contains different neuronal types which must function together as an organized network to optimize ventilation. We seek to relate the firing patterns of phrenic motoneurons and interneurons in the in vivo rat to respiratory pattern formation. We identify distinct subpopulations of inspiratory phrenic motoneurons, which exhibit highly similar firing dynamics within a frequency class, and identify a unique subset of these cells exhibiting tonic background firing during expiration. We relate the firing of inspiratory phrenic motoneurons with the highest firing frequencies to high-frequency oscillations in the phrenic nerve and demonstrate that this oscillatory behavior in the phrenic electroneurogram is not exclusively epiphenomenological, but rather, is directly underlied by the near-simultaneous firing of motoneurons at those frequencies. Additionally, we provide evidence for the existence of a fundamentally distinct group of cells - expiratory phrenic motoneurons - that fire exclusively or preferentially during expiration and discuss their potential physiological utility. Using phrenic nucleus microinjections of siRNA targeting the 65 and 67 kDa isoforms of glutamate decarboxylase, we show that local phrenic GABAergic interneurons play a critical role in respiratory pattern formation, contributing primarily to control of late-inspiratory and tonic expiratory activity. These findings suggest that local circuitry may play a more important function in shaping motor outputs than previously appreciated and that the phrenic nucleus plays an active role in respiratory pattern formation, as opposed to serving as a passive relay for descending brainstem respiratory inputs. We also show that crossed phrenic activity recovers acutely following a high cervical hemisection in the unanesthetized decerebrate rat. The sum total of the described work characterizes the role of phrenic motoneurons and interneurons and crossed bulbophrenic pathways in respiratory pattern formation during eupnea. While the pontomedullary network generates eupnea, evidence has emerged for phasic/rhythmic activity in respiratory-related discharge in various animals. We demonstrate that phasic activity can occur in phrenic nerve discharge of the unanesthetized decerebrate rat following high cervical transection (1) spontaneously, (2) as a consequence of asphyxia, and (3) in response to local treatment of C1-C2 spinal neurons with inhibitors of GABAA and glycinergic transmission. Spontaneous phrenic activity following spinalization included phasic discharge and slow oscillations suggesting the existence of intrinsic spinal generator mechanisms, which may underlie, and/or interact with, a putative spinal breathing generator. Asphyxia-induced phrenic bursting in spinalized animals suggests the existence of a spinal gasping generator. Bursting induced by treatment of C1-C2 spinal neurons with inhibitors of fast inhibitory synaptic transmission suggests the existence of intraspinal networks or intrinsic bursters capable of generating phasic discharge. In summary, rather than passively relaying pontomedullary eupnea to the diaphragm, the phrenic motor system is an active processing locus with distinct subpopulation of motomeurons and interneurons that contribute to eupneic pattern formation and possess the capacity to discharge phasically absent supraspinal drive.