Seven, Yasin B.

In skeletal muscles, force gradation depends on the level of motor unit (MU) activation. MU recruitment generally follows a specific order, with MUs comprising type I or type IIa fibers more active than type IIx and/or IIb. Direct evidence of MU recruitment across motor behaviors is limited to samples of relatively few MUs within a pool. The purpose of this study was to determine the extent of diaphragm MU recruitment across different motor behaviors. We hypothesized that in vivo labeling of individual neuromuscular junctions (NMJs) by FM4‐64 reflects the activation of MUs . Adult rats were exposed to eupneic (E), hypoxic/hypercarbic (H) conditions or supramaximal phrenic nerve stimulation (S). Individual diaphragm NMJs were type‐identified by myosin heavy chain expression. Average FM4‐64 labeling for NMJs at type I or IIa fibers vs. type IIx and/or IIb fibers increased progressively from E to H to S. The proportion of labeled NMJs of each type also increased significantly. At type I or IIa fibers, 40%, 59% and 90% of NMJs were labeled during E, H and S, respectively. At type IIx and/or IIb fibers, 21%, 32% and 82% of NMJs were labeled during E, H and S, respectively. Thus, using an in vivo labeling method there is direct evidence of progressive MU recruitment across different ventilatory and non‐ventilatory behaviors in the rat diaphragm. Supported by NIH grants HL37680 and AR51173, and the Mayo Clinic.

In previous studies, we showed that the root mean square of DIAm EMG correlates with transdiaphragmatic pressure (Pdi) reflecting force generation. We also showed that there is a shift in the power spectral density towards higher frequencies with recruitment of more fatigable fast‐twitch motor units. Recruitment of motor units leads to summation of motor unit action potentials (MUAP) causing non‐stationarity of the mean square value. Accordingly, non‐stationarity of EMG reflects motor unit recruitment. In the present study, we assessed stationarity of DIAm EMG across different ventilator and non‐ventilatory behaviors using a reverse arrangement test for 10‐ms binned mean square values. Stationarity was confirmed if 80% of the mean square calculations were within 2 standard deviations. In adult male rats, EMG recordings were obtained during eupnea, hypoxia‐hypercapnia (5% CO 2 /10% O 2 ), airway occlusion, and sneezing. At the onset of DIAm activity for each behavior, the EMG was stationary for ~75 ms. The EMG was also stationary for ~150 ms around the peak RMS for all motor behaviors. However, the period of non‐stationarity of DIAm EMG varied across motor behaviors: eupnea and hypoxia‐hypercapnia: 50 ms; airway occlusion: 240 ms; sneezing: 500 ms). Therefore, the duration of non‐stationarity of DIAm EMG increased with increasing force output; thus, reflecting the extent of motor unit recruitment.

Respiratory excitatory premotor drive from the medulla is widely distributed to phrenic motor neurons, and it is generally assumed that motor neuron recruitment order is determined by intrinsic, size‐related electrophysiological properties (Size Principle). During ventilatory behaviors, fatigue‐resistant diaphragm muscle (DIAm) motor units (smaller motor neurons) are recruited, but during more forceful non‐ventilatory behaviors such as airway occlusion, deep sighs and sneezing, additional more fatigable motor units are recruited. We hypothesized that a fixed recruitment order of DIAm motor units is maintained across different ventilatory and non‐ventilatory behaviors. Unitary motor unit discharge was recorded across eupnea, hypoxia‐hypercapnia (10% O 2 –5% CO 2 ), airway occlusion and deep sighs. The recruitment order displayed by motor units during eupnea and hypoxia‐hypercapnia was conserved during more forceful behaviors (82% of pairs maintained their recruitment order). Reversals of motor unit recruitment order occurred predominantly when the difference in onset times were within 20 ms. Onset discharge rates of motor units recruited during eupnea and hypoxia‐hypercapnia were lower than those of units recruited only during airway occlusion or deep sighs. These results support the Size Principle as an organizing principle of DIAm motor unit recruitment across a range of motor behaviors.

Motor units of the diaphragm muscle (DIAm) are recruited in an orderly fashion across ventilatory and non‐ventilatory tasks. Spinal hemisection at C2 (C2HS) blocks descending ipsilateral input to phrenic motoneurons and causes DIAm paralysis. With time after C2HS, there is spontaneous recovery of phrenic nerve activity ipsilateral to the injury. We hypothesized that recruitment order of DIAm motor units is maintained following recovery of DIAm electromyographic (EMG) activity post‐C2HS. DIAm EMG activity in adult male rats was recorded chronically using a pair of electrode wires implanted into the midcostal region bilaterally. DIAm EMG activity was strongly correlated to transdiaphragmatic pressure (Pdi) measured using esophageal and gastric balloons across different motor behaviors (Eupnea, hypoxia‐hypercapnia (10%O 2 and 5%CO 2 ), tracheal occlusion, fictive sneezing and bilateral supramaximal phrenic nerve stimulation). Fictive sneezing produced forces comparable to those elicited by phrenic nerve stimulation, and thus DIAm EMG activity during fictive sneezing was considered maximal. Absence of DIAm EMG activity was verified at the time of C2HS and 3 days (3D) post‐C2HS. Over time, a subset of animals displayed functional recovery of rhythmic DIAm EMG activity ipsilateral to C2HS, although DIAm EMG activity was reduced when compared to pre‐injury levels. EMG activity increased across ventilatory and non‐ventilatory tasks associated with increasing drive in C2HS animals displaying functional recovery, supporting a generalized strengthening of contralateral presynaptic drive over time following spinal cord injury.

Phrenic motor neurons are recruited across a range of motor behaviors to generate varying levels of diaphragm muscle (DIAm) force. We hypothesized that DIAm motor units are recruited in a fixed order across a range of motor behaviors of varying force levels, consistent with the Henneman Size Principle. Single motor unit action potentials and compound DIAm EMG activities were recorded in anesthetized, neurally intact rats across different motor behaviors, i.e., eupnea, hypoxia-hypercapnia (10% O 2 and 5% CO 2 ), deep breaths, sustained airway occlusion, and sneezing. Central drive [estimated by root-mean-squared (RMS) EMG value 75 ms after the onset of EMG activity (RMS 75 )], recruitment delay, and onset discharge frequencies were similar during eupnea and hypoxia-hypercapnia. Compared with eupnea, central drive increased (∼25%) during deep breaths, and motor units were recruited ∼12 ms earlier ( P < 0.01). During airway occlusion, central drive was ∼3 times greater, motor units were recruited ∼30 ms earlier ( P < 0.01), and motor unit onset discharge frequencies were significantly higher ( P < 0.01). Recruitment order of motor unit pairs observed during eupnea was maintained for 98%, 87%, and 84% of the same pairs recorded during hypoxia-hypercapnia, deep breaths, and airway occlusion, respectively. Reversals in motor unit recruitment order were observed primarily if motor unit pairs were recruited <20 ms apart. These results are consistent with DIAm motor unit recruitment order being determined primarily by the intrinsic size-dependent electrophysiological properties of phrenic motor neurons.

The diaphragm muscle (DIAm) is critically responsible for sustaining ventilation. Previously we showed in a commonly used model of spinal cord injury, unilateral spinal cord hemisection at C 2 (SH), that there are minimal changes to muscle fiber cross-sectional area (CSA) and fiber type distribution following 14 days of SH-induced ipsilateral DIAm inactivity. In the present study, effects of long-term SH-induced inactivity on DIAm fiber size and force were examined. We hypothesized that prolonged inactivity would not result in substantial DIAm atrophy or force loss. Adult rats were randomized to control or SH groups ( n = 34 total). Chronic bilateral DIAm electromyographic (EMG) activity was monitored during resting breathing. Minimal levels of spontaneous recovery of ipsilateral DIAm EMG activity were evident in 42% of SH rats (<25% of preinjury root mean square amplitude). Following 42 days of SH, DIAm specific force was reduced 39%. There was no difference in CSA for type I or IIa DIAm fibers in SH rats compared with age, weight-matched controls (classification based on myosin heavy chain isoform expression). Type IIx and/or IIb DIAm fibers displayed a modest 20% reduction in CSA ( P < 0.05). Overall, there were no differences in the distribution of fiber types or the contribution of each fiber type to the total DIAm CSA. These data indicate that reduced specific force following prolonged inactivity of the DIAm is associated with modest, fiber type selective adaptations in muscle fiber size and fiber type distribution.