Recruitment of rat diaphragm motor units across motor behaviors with different levels of diaphragm activation Yasin B. Seven, Carlos B. Mantilla and Gary C. Sieck
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J Appl Physiol 117: 1308–1316, 2014. First published September 25, 2014; doi:10.1152/japplphysiol.01395.2013.
Recruitment of rat diaphragm motor units across motor behaviors with different levels of diaphragm activation Yasin B. Seven,1 Carlos B. Mantilla,1,2 and Gary C. Sieck1,2 1
Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, Minnesota; and 2Department of Anesthesiology, Mayo Clinic, Rochester, Minnesota
Submitted 2 January 2014; accepted in final form 21 September 2014
motor unit recruitment order; recruitment reversal; discharge rates; doublets; electromyography; neuromotor control IN SKELETAL MUSCLES,
increasing levels of force are accomplished by recruiting additional motor units and/or by increasing the discharge frequencies of recruited motor units (1). Based on studying single motor neuron axons in ventral root filaments in spinalized cat, Henneman and colleagues (16, 27, 43) showed that motor units are recruited in a fixed order based on the size-dependent electrophysiological properties (e.g., conduction velocity) of motor neurons. The resulting Henneman Size Principle is now generally accepted and has been demonstrated in numerous skeletal muscles including the diaphragm muscle (DIAm) and across different species including humans (13, 29, 38, 39, 44). Dick and colleagues (13) clearly demonstrated the Size Principle in the cat DIAm but only during hypercapnia in anesthetized and mechanically ventilated animals. Address for reprint requests and other correspondence: G. C. Sieck, Dept. of Physiology and Biomedical Engineering, Mayo Clinic College of Medicine, 200 First St. SW, Rochester, MN 55905 (e-mail: [email protected]). 1308
Several studies have evaluated DIAm motor unit recruitment and discharge rate during eupnea and hypoxia-hypercapnia (7, 8, 14, 32, 35). Generally, these studies found that when ventilation was stimulated by hypoxia and/or hypercapnia, additional motor units were recruited and the peak discharge rates of motor units increased. Butler et al. (8) reported in the human DIAm that motor unit recruitment order across different voluntary tasks was generally fixed with consistent lung volume-related thresholds. However, they reported that reversals of motor unit recruitment order sometimes occurred during voluntary tasks. Voluntary activation of the DIAm is very likely to involve different neuromotor control pathways that may affect recruitment order. Furthermore, although these previous studies examined DIAm motor unit recruitment and discharge frequency during voluntary tasks that accomplished different lung volumes and inspiratory flow rates, they did not directly assess DIAm motor unit recruitment order during motor behaviors of increasing force generation. Compared with eupnea and hypoxia-hypercapnia, greater levels of DIAm force are generated during deep breaths, airway occlusion, and sneezing (25, 34). The goal of this study was to assess the pattern of DIAm motor unit recruitment across motor behaviors that result in increasing force generation. 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. METHODS
Animals. Fifteen adult, male Sprague-Dawley rats weighing 320 – 340 g were used for this study. All experimental procedures and techniques were approved by the Mayo Clinic Institutional Animal Care and Use Committee and were in accordance with the American Physiological Society Animal Care Guidelines. An intramuscular injection of ketamine (90 mg/kg) and xylazine (10 mg/kg) was administered during all experimental procedures. Motor behaviors. Both single motor unit and compound DIAm EMG were recorded during: 1) eupnea (breathing in room air), 2) hypoxia-hypercapnia (breathing in air with 10% O2 and 5% CO2 in N2), 3) deep breath [spontaneous “sighs”; root-mean-squared (RMS) EMG peak ⬎ 2 times eupneic RMS EMG peak value], 4) airway occlusion (sustained closure of airways for ⬃40 s), and 5) sneezing (induced by intranasal administration of capsaicin) as described previously (24, 25, 33). All measurements were made with the animal in supine position with body temperature maintained at 37°C using a heating pad. In analyses of DIAm EMG activity during eupnea and hypoxia-hypercapnia, a 30-s period was used excluding spontaneous deep breaths. During airway occlusion, efforts generated in the last 5-s period of occlusion (4 – 6 efforts) were analyzed. Animals recovered for at least 5 min after each behavior to allow values to return to baseline.
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Seven YB, Mantilla CB, Sieck GC. Recruitment of rat diaphragm motor units across motor behaviors with different levels of diaphragm activation. J Appl Physiol 117: 1308 –1316, 2014. First published September 25, 2014; doi:10.1152/japplphysiol.01395.2013.—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% O2 and 5% CO2), deep breaths, sustained airway occlusion, and sneezing. Central drive [estimated by rootmean-squared (RMS) EMG value 75 ms after the onset of EMG activity (RMS75)], 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.
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rates. Double discharges (instantaneous discharge rates ⬎ 100 Hz) were excluded from the analysis of discharge rates. Statistical analysis. Parameters calculated for each burst were averaged across multiple bursts for each motor behavior. The values obtained from the same motor unit across different motor behaviors were treated as repeated measures. Statistics of multiple units were summarized as means ⫾ SE. In the statistical analyses, repeatedmeasures univariate F-test with Greenhouse-Geisser adjustment was used (31, 40). In other comparisons of motor unit discharge properties (e.g., onset and peak motor unit discharge frequencies) across motor behaviors, only those units displaying activity across all behaviors were compared using one-way ANOVA. When F-tests showed significant differences, Tukey-Kramer Honestly Significant Difference test was used to analyze differences post hoc. JMP 9.0 was used to perform statistical calculations (SAS Institute, Cary, NC). Significance level was set to P ⬍ 0.05. RESULTS
Compound DIAm EMG across motor behaviors. Consistent with our previous findings (24, 25, 33), the maximal RMS EMG value (RMSmax) was observed during sneezing (Fig. 1). Thus all RMS values were normalized to RMSmax during sneezing. Representative traces for single RMS EMG bursts across different motor behaviors of the DIAm are shown in Fig. 2A. To estimate neural drive to phrenic motor neurons, the RMS value of the compound DIAm EMG was measured at 75 ms (RMS75) after the onset of activity (Fig. 2B). The RMS75 values during eupnea (15 ⫾ 2%) and hypoxia-hypercapnia (18 ⫾ 2%) were comparable. A slight but significant increase in RMS75 was observed during deep breaths (19 ⫾ 3%; P ⬍ 0.05) compared with eupnea. Importantly, the increase in RMS75 during airway occlusion (42 ⫾ 5%; P ⬍ 0.001) was substantially greater compared with all other motor behaviors. During sneezing, there was a multiphasic pattern, with a relatively low and variable (within and across animals) level of activity at the initiation of a sneeze (for ⬃120 ms) followed by a augmenting increase in RMS DIAm EMG activity that followed a trajectory similar to deep breaths. Finally, there was a third phase with steeper increase in RMS EMG activity. Due
Fig. 1. Representative compound diaphragm EMG recordings and root-mean-squared (RMS) EMG tracings across eupnea, hypoxia-hypercapnia (10% O2, 5% CO2), spontaneous deep breath (sigh), airway occlusion, and sneezing. The main response to hypoxia-hypercapnia was increased respiratory rate (reduced expiratory time). The amplitude of diaphragm EMG activity, as measured by RMS EMG, increased significantly during spontaneous deep breaths (sigh), airway occlusion, and sneezing compared with eupnea and hypoxiahypercapnia.
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Single motor unit and compound DIAm EMG recordings. Single unit and compound DIAm EMG were simultaneously recorded using different electrode and acquisition configurations. To record compound EMG signal, two pairs of teflon-coated, multistranded stainless-steel wires (AS631, Cooner Wire, Chatsworth, CA) were implanted bilaterally into the midcostal DIAm following laparotomy. Insulation was removed for ⬃2 mm at the point of juxtaposition with the DIAm. The compound EMG signal was differentially amplified (2,000⫻), bandpass filtered (20 –1,000 Hz) using an analog amplifier (model 2124, DATA), and digitally sampled at a frequency of 2 kHz using a data-acquisition board (National Instruments, Austin, TX) controlled by a custom-made program (LabView 8.2; National Instruments, Austin, TX). Root-mean squared (RMS) EMG signal was computed from the compound EMG signal using a 50-ms window as described previously (23–25, 33, 34). For single motor unit recordings, each pair of insulated fine-wire electrodes (40 m diameter; Moleculoy, Bridgeport Insulated Wire, Bridgeport, CT) was inserted into the midcostal DIAm. Single motor unit EMG signals were differentially amplified (2,000⫻), bandpass filtered (100 –5,000 Hz) using an analog amplifier (Model EMG100C, Biopac Systems, Goleta, CA), and digitally sampled at a frequency of 20 kHz. Single motor unit action potentials (MUAPs) were identified by a custom thresholding and template-matching algorithm in MATLAB (Mathworks, Natick, MA). Recruitment delay of each motor unit was calculated as the time difference between compound EMG burst onset and the first MUAP of each motor unit within the burst. The average recruitment delay of each motor unit was determined by averaging multiple bursts for each motor behavior. The onset of compound EMG was set as the point where rectified compound EMG value was twice the baseline value. Recruitment order of motor units was determined by analyzing motor unit pairs separately across motor behaviors. The difference in recruitment delays of individual motor units in a pair was evaluated. Recruitment order of a pair of motor units was considered to be reversed if a motor unit in the pair was recruited earlier than the other unit during eupnea but later than the other unit during another motor behavior. The relationships between differences in recruitment delays and the percentage of reversals were also evaluated. Onset and peak discharge rates were calculated as the reciprocal of interspike interval for each motor unit across motor behaviors. Instantaneous discharge rates were filtered with a two-point moving average window to reduce the effects of spontaneous changes in discharge
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to variability at the onset of EMG activity, the RMS75 during sneezing was highly variable and did not reflect the final extent of DIAm EMG activation. Peak RMS EMG activity of the DIAm was 28 ⫾ 3% during eupnea, 37 ⫾ 3% during hypoxia-hypercapnia (P ⬍ 0.01 compared with eupnea), 69 ⫾ 4% during deep breaths (P ⬍ 0.001 compared with eupnea), 62 ⫾ 3% during airway occlusion (P ⬍ 0.001 compared with eupnea), and maximum during sneezing (100%; P ⬍ 0.001 compared with eupnea) (Fig. 2B). The peak RMS DIAm EMG during airway occlusion was comparable to that during deep breaths (P ⫽ 0.89), but peak
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RMS EMG during airway occlusion and deep breaths were significantly greater than hypoxia-hypercapnia (P ⬍ 0.001). DIAm motor unit recruitment across motor behaviors. Figure 3 displays representative compound and single motor unit DIAm EMG activities during a eupneic inspiratory effort. The activities of three motor units were discriminated based on waveform analyses that showed consistent patterns across multiple (⬎10) discharges. In this example, DIAm motor units were recruited in an orderly fashion during the first 100 ms of the eupneic EMG burst duration. Additional motor units contributed to the compound DIAm EMG, but the waveforms of these motor units could not be clearly discriminated across eupneic bursts. After recruitment, motor unit discharge rate progressively increased with peak discharge frequency attained at approximately midway through the effort. In this example, the onset discharge rates were 32, 33, and 29 Hz for motor units 1, 2, and 3, respectively. Peak discharge rates were 48, 47, and 52 Hz, respectively. Most motor units recruited during eupnea could also be discriminated during more forceful DIAm motor behaviors. Motor unit discrimination was more reliable during eupnea and hypoxia-hypercapnia, but during airway occlusion and deep breaths, fewer of those motor units could be discriminated around the peak RMS EMG activity due to amplitude summation/cancellation of additionally recruited motor units. A total of 98 motor units were discriminated across all motor behaviors. Table 1 shows the number of motor units discriminated during each motor behavior and during which behavior those motor units were recruited first. For example, during eupnea 64 motor units were recorded and all of these units were first recruited during eupnea. Of these 64 motor units, the discharge of 55, 48, and 40 of the same motor units could be discriminated during hypoxia-hypercapnia, deep breaths, and airway occlusion, respectively. The number of motor units was decreased, because we were unable to discriminate individual motor units due to amplitude summation/cancellation of multiple motor unit action potentials during more forceful motor behaviors. During hypoxia-hypercapnia, in addition to the 55
Fig. 3. Representative compound DIAm EMG burst and simultaneous single motor unit recordings obtained during eupnea in a spontaneously breathing anesthetized rat. The arrow at top indicates the onset point of compound EMG activity. Three motor units were discriminated via waveform analysis throughout the EMG burst with the superimposed signal shown at left and trains of motor unit action potentials at right for each motor unit. Note differences in time scales between superimposed single motor unit waveforms and compound EMG recordings.
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Fig. 2. Central drive to the diaphragm muscle (DIAm), as measured by RMS DIAm EMG at the initial 75 ms (RMS75), and peak RMS EMG normalized to the maximal RMS EMG value observed during sneezing. A: representative RMS EMG traces averaged within an animal across motor behaviors. Dashed line at 75 ms indicates the point at which RMS75 was calculated (see METHODS for details). B: normalized RMS75 (open bars) and peak RMS EMG (black bars) across DIAm motor behaviors (expressed as % of maximum RMS EMG displayed during sneezing). RMS75 was comparable during eupnea and hypoxia-hypercapnia. Deep breaths presented higher RMS75 value compared with eupnea (P ⬍ 0.05). RMS75 was substantially higher during airway occlusion (P ⬍ 0.001). Peak RMS EMG progressively increased from eupnea to hypoxia-hypercapnia (P ⬍ 0.01) and to deep breath (P ⬍ 0.001). During airway occlusion, peak RMS EMG was comparable to deep breaths (P ⬎ 0.05) but greater than eupnea (P ⬍ 0.001) and hypoxia-hypercapnia (P ⬍ 0.001). *P ⬍ 0.05 vs. eupnea; #P ⬍ 0.05 vs. hypoxia-hypercapnia; &P ⬍ 0.05 vs. deep breaths; §P ⬍ 0.05 vs. RMS75 for same behavior.
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Table 1. Number of motor units recorded across motor behaviors Number of Motor Units Discriminated During Motor Units First Recruited During
Eupnea Hypoxia-hypercapnia Deep breath Airway occlusion Total
Deep breath
Airway occlusion
55 8
48 4 10
40 4
63
62
Eupnea
Hypoxia-hypercapnia
64
64
16 60
Motor units are segregated according to the motor behavior during which they were first recruited.
Fig. 4. Recruitment delays of motor units discriminated across eupnea, hypoxia-hypercapnia, deep breath, and airway occlusion. Motor units were recruited earlier during deep breaths compared with eupnea (P ⬍ 0.01). During airway occlusion, motor units were recruited very early compared with all other motor behaviors (P ⬍ 0.01). *P ⬍ 0.05 vs. eupnea; #P ⬍ 0.05 vs. hypoxiahypercapnia; &P ⬍ 0.05 vs. deep breaths.
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motor units recruited during eupnea, eight new units were discriminated that had not been active previously during eupnea. Of these eight motor units freshly recruited during hypoxia-hypercapnia, the activity of only four units could still be distinguished during deep breaths and airway occlusion. During deep breaths, the activity of 10 additional motor units could be discriminated beyond those that were recruited during eupnea and hypoxia-hypercapnia. During airway occlusion, an additional 16 motor units were freshly recruited. Figure 4 summarizes the recruitment delays of 40 DIAm motor units that were discriminated across all motor behaviors. Recruitment delays were 43.0 ⫾ 5.0 ms during eupnea, 35.0 ⫾ 4.5 ms during hypoxia-hypercapnia, 31.5 ⫾ 4.0 ms during deep breaths, and 14.5 ⫾ 2.0 ms during airway occlusion. These differences in recruitment delays generally corresponded with differences in estimated neural drive to phrenic motor neurons (RMS75; Fig. 2). Compared with eupnea, the recruitment delay during deep breaths was significantly shorter (P ⬍ 0.01). However, the greatest shortening of motor unit recruitment delay was observed during airway occlusion compared with all other motor behaviors (P ⬍ 0.01). Figure 5 shows scatterplots of recruitment delays of the same motor units during eupnea compared with other motor
Fig. 5. Scatterplots displaying recruitment delays of individual motor units during eupnea vs. hypoxia-hypercapnia (A), deep breaths (B), and airway occlusion (C). The identity line is shown in gray. Most motor units were recruited earlier during other motor behaviors compared with eupnea (i.e., lower right of the identity line). Earlier recruitment of motor units was more evident during airway occlusion (shallower slope; m ⫽ 0.37). The r2 values of the fitted lines were 0.47, 0.74, and 0.35 during eupnea vs. hypoxia-hypercapnia, deep breaths, and airway occlusion, respectively. Diaphragm motor units recruited very early (⬍25 ms) during eupnea were recruited both earlier and later during other motor behaviors. However, the motor units with recruitment delays ⬎25 ms during eupnea were almost always recruited earlier during other motor behaviors.
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Fig. 6. Distribution of difference in recruitment delay of motor unit pairs across motor behaviors. The difference in recruitment delay during eupnea is considered as the standard (assumed as positive value) for a motor unit pair and is indicated in the x-axis. An average difference in recruitment delay becoming negative during another motor behavior indicates that a reversal of motor unit recruitment order has occurred. A: motor unit pairs with conserved (i.e., maintained) recruitment order across all motor behaviors. B: motor unit pairs with reversed recruitment order during any motor behavior compared with eupnea. Most recruitment reversals occurred for motor unit pairs with a difference in recruitment delay during eupnea of ⬍20 ms.
DIAm motor unit discharge rates across motor behaviors. Once recruited, the discharge rates of DIAm motor units displayed an augmenting pattern until peak discharge rates were achieved. Figure 7 compares the onset and peak discharge rates of motor units across all motor behaviors. At the onset, the firing frequencies were comparable during eupnea (27 ⫾ 2 Hz), hypoxia-hypercapnia (30 ⫾ 2 Hz), and deep breath (29 ⫾ 2 Hz). However, during airway occlusion, onset firing frequencies were significantly higher compared with all other behaviors (37 ⫾ 3 Hz; P ⬍ 0.01). Higher onset firing frequencies during airway occlusion might be due to increased drive at the onset of the behavior. Occasionally, a short discharge interval (or doublets defined as consecutive MUAPs with instantaneous discharge frequency ⬎100 Hz) was observed at the onset of airway occlusion (Fig. 8). In the example shown
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behaviors. The identity line (shown in gray) indicates the points where a motor unit is recruited with the same delay during eupnea and the other motor behavior being compared. When a motor unit is recruited later during eupnea but earlier during another motor behavior, it is shown in the lower right of the identity line. According to Fig. 5, most of the motor units were recruited earlier during other motor behaviors compared with eupnea, but the extent of deviation from the line of identity varied across motor behaviors. In the graph presented in Fig. 5, slope values lower than unity indicate earlier recruitment of motor units compared with eupnea. Earlier recruitment of motor units was evident across all motor behaviors compared with eupnea (i.e., all slope values ⬍ 1.00). During hypoxia-hypercapnia and deep breaths the slope was 0.64 and 0.72 with r2 values of 0.47 and 0.74, respectively. Earlier recruitment of motor units was most pronounced during airway occlusion as shown by a shallower slope (m ⫽ 0.37, r2 ⫽ 0.35; Fig. 5C). In general, those motor units recruited very early (⬍25 ms) during eupnea were also recruited earlier across other motor behaviors, and in some cases recruitment delays became longer compared with eupnea (above the line of identity; Fig. 5). However, for those motor units with recruitment delays ⬎25 ms during eupnea, they were almost always recruited earlier during other motor behaviors (below the line of identity; Fig. 5). There were eight motor units that were additionally recruited during hypoxia-hypercapnia. These motor units had recruitment delays ranging from 40 to 330 ms (average recruitment delay of 96.7 ⫾ 37.0 ms). During deep breaths, an additional 10 motor units were recruited with recruitment delays ranging from 30 to 550 ms (average recruitment delay of 232.9 ⫾ 41.1 ms). During airway occlusion, an additional 16 motor units were recruited with recruitment delays ranging from 20 to 360 ms (average recruitment delay of 160.2 ⫾ 27.7 ms). Recruitment order of DIAm motor unit pairs across motor behaviors. In addition to single DIAm motor unit analyses, motor units were also analyzed in pairs across more than one motor behavior, and the relative timing of motor unit recruitment could be compared. For example, one motor unit pair was analyzed if two motor units were discriminated. Three motor unit pairs were analyzed for three motor units. Six motor unit pairs were analyzed for four motor units. A total of 104 pairs of motor units were initially discriminated during eupnea and subsequently followed across multiple motor behaviors. Compared with eupnea, the recruitment order of DIAm motor units was conserved for 98%, 87%, and 84% of the motor unit pairs during hypoxia-hypercapnia, deep breath, and airway occlusion, respectively. Figure 6A shows a histogram of differences in recruitment delays between pairs of motor units during eupnea. Although the order of recruitment was generally conserved across motor behaviors, reversals of recruitment order did occur in 2–16% of motor unit pairs depending on motor behavior. In most of the motor unit pairs in which reversal of recruitment order was observed (9 of 13 motor unit pairs), initial differences in recruitment delays during eupnea were ⬍20 ms (Fig. 6B). Among all motor unit pairs, 20% of those with initial differences in recruitment delays during eupnea ⬍20 ms displayed reversal, while for those units with initial differences in recruitment delays during eupnea ⬎20 ms, only 8% showed reversal during another motor behavior.
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DISCUSSION
in Fig. 8, the doublet discharge interval was 9.4 ms (106 Hz) with a sustained discharge rate of 40 –50 Hz. Doublet discharge of motor units was not observed in any other motor behavior in which motor unit discharge could be discriminated. Peak firing frequencies of DIAm motor units were slightly higher during hypoxia-hypercapnia (43 ⫾ 3 Hz) compared with eupnea (37 ⫾ 3 Hz; P ⬍ 0.05, Fig. 7). Peak firing frequencies during deep breaths (61 ⫾ 8 Hz) and airway occlusion (56 ⫾ 7 Hz) were comparable, but higher compared with both eupnea (P ⬍ 0.01) and hypoxia-hypercapnia (P ⬍ 0.01) (Fig. 7).
Fig. 8. Representative single motor unit recordings obtained using fine wire electrodes in the DIAm across motor behaviors (left). Waveform analysis was used to identify the motor unit with the averaged signal and instantaneous discharge frequency (right) shown for this motor unit across behaviors. Notice the doublet discharge (*; discharge frequency ⬎ 100 Hz) at the beginning of airway occlusion.
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Fig. 7. Onset (open bars) and peak (black bars) discharge frequencies across motor behaviors. Onset discharge frequencies of DIAm motor units were greater during airway occlusion compared with eupnea, hypoxia-hypercapnia, and deep breaths (P ⬍ 0.01). Peak discharge rates were significantly higher than onset discharge rates regardless of the motor behavior (P ⬍ 0.01). Peak discharge frequencies of motor units during deep breath and airway occlusion were higher than eupnea and hypoxia-hypercapnia (P ⬍ 0.01). Hypoxiahypercapnia resulted in higher peak discharge frequencies than eupnea (P ⬍ 0.01). *P ⬍ 0.05 vs. eupnea; #P ⬍ 0.05 vs. hypoxia-hypercapnia; &P ⬍ 0.05 vs. deep breaths; §P ⬍ 0.05 vs. RMS75 for same behavior.
The results of the present study support our hypothesis that DIAm motor units are recruited in a fixed order across motor behaviors of varying force, consistent with intrinsic sizerelated electrophysiological properties of phrenic motor neurons (i.e., the Henneman Size Principle). Central drive to phrenic motor neurons (indexed by RMS75 DIAm EMG) was lowest during eupnea and hypoxia/hypercapnia compared with deep breaths and airway occlusion. In particular, central drive was substantially increased during airway occlusion compared with all other motor behaviors. Consistent with this escalation of central drive, single motor unit recruitment delays were shortest during airway occlusion. The motor pattern during sneezing appeared to be multiphasic, with central drive being relatively low and variable during the initial ⬃120 ms, likely reflecting the more complex activation of inspiratory and expiratory muscles in this behavior. Recruitment delays across pairs of simultaneously recorded DIAm motor units were used to determine the order of motor unit recruitment across motor behaviors with different levels of force generation [as indicated by the RMS DIAm EMG (25, 34)]. For these comparisons, motor unit recruitment order was indexed to that observed during eupnea. Accordingly, changes in motor unit recruitment order during different motor behaviors were analyzed only for those motor unit pairs initially recorded during eupnea. However, with increasing central drive in deep breaths and airway occlusion, the number of motor units recruited increased and recruitment delays were shortened. Regardless, compared with eupnea, motor unit recruitment order was mostly maintained (⬎80% of all motor unit pairs across all behaviors). Motor unit pairs displayed reversals of recruitment order primarily when recruitment delays differed by ⬍20 ms in eupnea. Central drive to phrenic motor neurons. The extent of central drive to phrenic motor neurons cannot be directly measured. However, central drive has been estimated by using a number of outcome measures. Previously, we demonstrated that DIAm RMS EMG amplitude is highly correlated with transdiaphragmatic pressure (Pdi; an estimate of DIAm force; r2 ⫽ 0.78) across motor behaviors with different levels of force when the abdomen is bound (25, 34). In the present study, it was not possible to bind the abdomen since fine-wire elec-
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recruitment delays are shortened during motor behaviors associated with more rapid development of force, e.g., increased inspiratory flow rate, airway occlusion, or airway clearance (coughing or vomiting). In a recent study, earlier recruitment of DIAm motor units was confirmed by evaluating the stationarity of DIAm EMG signal, in which changes in EMG mean-square values were assessed by the reverse arrangement test (33). The period of nonstationarity at the onset of the EMG burst (typically ⬍75 ms; hence the selection of the RMS75 measure) correlated well with the period of motor unit recruitment, with both recruitment delays and the period of nonstationarity shortening from eupnea to hypoxia-hypercapnia and to airway occlusion. Motor unit recruitment order is conserved across different force levels. Phrenic motor neurons are thought to receive distributed input from the medulla via premotor neurons (9, 10). Differences in phrenic motoneuron somal and dendritic surface areas (30) likely contribute to orderly recruitment (15, 16, 27, 43) based on their size-dependent electrophysiological properties. Indeed, phrenic motor neurons with slower axonal conduction velocities (smaller motor neurons) are recruited earlier than those with faster conduction velocities (larger motor neurons) (13) in spontaneously-breathing anesthetized cats. Previous studies examining DIAm motor unit activities during different motor behaviors (3, 6, 19, 20, 28, 36) did not compare recruitment order of motor unit pairs in neurally intact animals across motor behaviors requiring substantially different levels of force generation. In the present study, the order of DIAm motor unit recruitment established during eupnea was indeed maintained for 98%, 87%, and 84% of motor unit pairs during hypoxia-hypercapnia, deep breaths, and airway occlusion, respectively. With increasing drive, earlier recruitment of all active motor units is consistent with the slight reduction in the maintenance of motor unit recruitment order. In paralyzed, mechanically ventilated, and decerebrate cats, no reversals were observed in 24 filaments with recordings of motor neuron pairs during fictive coughing, but two of nine motor neuron pairs displayed spontaneous reversals during fictive vomiting (28). It is worth noting that in the present study for cases where DIAm motor unit recruitment order was reversed across behaviors, the difference in recruitment delays during eupnea was very short (20 ms or less). Thus loss of a strict order of motor unit recruitment is consistent with “noise” in neural activation occurring for motor unit pairs with similar electrophysiological properties (37). Taken together, these results are consistent with widely distributed input to phrenic motor neurons and the order of phrenic motor neurons recruitment being based on their size-related electrophysiological properties. Furthermore, these results do not support selective input to phrenic motor neurons emanating from central pattern generators for distinct motor behaviors. In other words, these motor behaviors are distinct in terms of the degree of activation, but not necessarily in other ways. Motor unit discharge rates. All motor behaviors in skeletal muscles are accomplished by controlling both recruitment and discharge frequencies of motor units (1). The onset discharge rates of DIAm motor units were comparable across motor behaviors with the exception of airway occlusion where onset discharge rates were higher. The higher motor unit discharge rate during airway occlusion reflects a greater central drive as
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trodes were dislocated from the DIAm especially during more forceful motor behaviors. Despite the abdomen not being bound in the present study, peak RMS EMG values and the rate of rise of RMS EMG were comparable to those previously reported in rats (25). However, RMS EMG is also influenced by the duration of the inspiratory effort (modulated by lung stretch receptor feedback). Accordingly, DIAm RMS EMG 75 ms after onset (RMS75) was used as a measure of central drive in the present study. Of note, the RMS75 value was very consistent across DIAm EMG bursts within each motor behavior for each animal (coefficient of variation: ⬃10%). The DIAm RMS75 EMG was similar during eupnea and hypoxia-hypercapnia. Previous studies are generally in agreement with a main effect of hypoxia (10% O2)-hypercapnia (5% CO2) on respiratory frequency rather than an increase in peak DIAm EMG activity (and thus tidal volume) in anesthetized rats (24, 25, 33, 34) and paralyzed, mechanically ventilated and decerebrate cats (17). In conscious humans in hyperoxic conditions, exposure to 7% CO2 increased DIAm EMG activity by ⬃20% (slope of the rectified moving average) following a 10-mmHg increase in arterial CO2 (22). Consistently, mouth occlusion pressure at 100 ms after the onset of inspiratory activity (P100) increased from 3 to 7 cmH2O following a 10-mmHg increase in arterial CO2 (2, 21, 42). These discrepancies in the response to hypoxia-hypercapnia may relate to species, anesthetic effects, choice of preparations (decerebrate vs. intact), selection of hypercapnic and/or hypoxic conditions, as well as the method for assessment. Regardless, the change in central drive to phrenic motor neurons during hypoxia-hypercapnia was far from maximal. DIAm motor unit recruitment across motor behaviors of different force levels. Theoretical models of rat DIAm motor unit recruitment suggest that a significant number of DIAm motor units (at least 45–50%) are not recruited during eupnea (25, 26). In general agreement, additional motor units were recruited with increasing central drive compared with eupnea. Unfortunately, limitations related to the discrimination of single motor unit action potentials and volume conduction preclude estimations of the proportion of motor units recruited during each motor behavior and result in fewer motor units being discriminated during more forceful behaviors. The delay in motor unit recruitment for an active motor unit reflects changes in central drive as well as intrinsic, size-related electrophysiological properties of the motor neurons. Phrenic motor neurons receive descending neural drive with time differences on the order of only a few milliseconds (5, 9, 10, 41), at least during eupnea. In the present study, recruitment delays of DIAm motor units varied between 0 and 150 ms during eupnea (43 ms on average). Thus shortened recruitment delays during deep breaths (32 ms) and airway occlusion (15 ms) are consistent with increased central drive during more forceful motor behaviors. These findings are generally consistent with previous studies reporting reduced motor unit recruitment delays in paralyzed, vagotomized, and mechanically ventilated rats (20) and cats (18, 36). These results are also consistent with the observation of Butler et al. (8) for recruitment of DIAm motor units in humans during voluntary tasks. They found that when inspiratory flow increased (perhaps reflecting an increased drive to breathe), motor units were recruited earlier although lung volume threshold remained constant. Overall, these results indicate that DIAm motor unit
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GRANTS This work was supported by National Heart, Lung, and Blood Institute Grant HL-096750 and the Mayo Clinic. DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the author(s). AUTHOR CONTRIBUTIONS Author contributions: Y.B.S., C.B.M., and G.C.S. conception and design of research; Y.B.S. performed experiments; Y.B.S. and G.C.S. analyzed data; Y.B.S., C.B.M., and G.C.S. interpreted results of experiments; Y.B.S., C.B.M., and G.C.S. prepared figures; Y.B.S., C.B.M., and G.C.S. drafted manuscript; Y.B.S., C.B.M., and G.C.S. edited and revised manuscript; Y.B.S., C.B.M., and G.C.S. approved final version of manuscript. REFERENCES 1. Adrian ED, Bronk DW. The discharge of impulses in motor nerve fibres. II. The frequency of discharge in reflex and voluntary contractions. J Physiol 67: i3–151, 1929. 2. Altose MD, Kelsen SG, Stanley NN, Levinson RS, Cherniack NS, Fishman AP. Effects of hypercapnia on mouth pressure during airway occlusion in conscious man. J Appl Physiol 40: 338 –344, 1976. 3. Arita H, Bishop B. Firing profile of diaphragm single motor unit during hypercapnia and airway occlusion. J Appl Physiol 55: 1203–1210, 1983. 4. Bawa P, Calancie B. Repetitive doublets in human flexor carpi radialis muscle. J Physiol 339: 123–132, 1983. 5. Berger AJ. Phrenic motoneurons in the cat: subpopulations and nature of respiratory drive potentials. J Neurophysiol 42: 76 –90, 1979. 6. Bishop B, Settle S, Hirsch J. Single motor unit activity in the diaphragm of cat during pressure breathing. J Appl Physiol 50: 348 –357, 1981. 7. Butler JE. Drive to the human respiratory muscles. Respir Physiol Neurobiol 159: 115–126, 2007.
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8. Butler JE, McKenzie DK, Gandevia SC. Discharge properties and recruitment of human diaphragmatic motor units during voluntary inspiratory tasks. J Physiol 518: 907–920, 1999. 9. Cohen MI, Piercey MF, Gootman PM, Wolotosky P. Synaptic connections between medullary inspiratory neurons and phrenic motoneurons as revealed by cross-correlation. Brain Res 81: 319 –324, 1974. 10. Davies JG, Kirkwood PA, Sears TA. The distribution of monosynaptic connexions from inspiratory bulbospinal neurones to inspiratory motoneurones in the cat. J Physiol 368: 63–87, 1985. 11. Desmedt JE, Godaux E. Ballistic contractions in fast or slow human muscles: discharge patterns of single motor units. J Physiol 285: 185–196, 1978. 12. Desmedt JE, Godaux E. Ballistic contractions in man: characteristic recruitment pattern of single motor units of the tibialis anterior muscle. J Physiol 264: 673–693, 1977. 13. Dick TE, Kong FJ, Berger AJ. Correlation of recruitment order with axonal conduction velocity for supraspinally driven diaphragmatic motor units. J Neurophysiol 57: 245–259, 1987. 14. Gandevia SC, Leeper JB, McKenzie DK, De Troyer A. Discharge frequencies of parasternal intercostal and scalene motor units during breathing in normal and COPD subjects. Am J Respir Crit Care Med 153: 622–628, 1996. 15. Henneman E. Relation between size of neurons and their susceptibility to discharge. Science 126: 1345–1346, 1957. 16. Henneman E, Somjen G, Carpenter DO. Functional significance of cell size in spinal motoneurons. J Neurophysiol 28: 560 –580, 1965. 17. Hwang JC, St. John WM. Facilitation and inhibition of phrenic motoneuronal activities by lung inflation. J Appl Physiol 74: 2485–2492, 1993. 18. Iscoe S, Dankoff J, Migicovsky R, Polosa C. Recruitment and discharge frequency of phrenic motoneurones during inspiration. Respir Physiol Neurobiol 26: 113–128, 1976. 19. Jodkowski JS, Viana F, Dick TE, Berger AJ. Electrical properties of phrenic motoneurons in the cat: correlation with inspiratory drive. J Neurophysiol 58: 105–124, 1987. 20. Kong FJ, Berger AJ. Firing properties and hypercapnic responses of single phrenic motor axons in the rat. J Appl Physiol 61: 1999 –2004, 1986. 21. Lederer DH, Altose MD, Kelsen SG, Cherniack NS. Comparison of occlusion pressure and ventilatory responses. Thorax 32: 212–220, 1977. 22. Lopata M, Evanich MJ, Lourenco RV. Quantification of diaphragmatic EMG response to CO2 rebreathing in humans. J Appl Physiol 43: 262– 270, 1977. 23. Mantilla CB, Greising SM, Zhan WZ, Seven YB, Sieck GC. Prolonged C2 spinal hemisection-induced inactivity reduces diaphragm muscle specific force with modest, selective atrophy of type IIx and/or IIb fibers. J Appl Physiol 114: 380 –386, 2013. 24. Mantilla CB, Seven YB, Hurtado-Palomino JN, Zhan WZ, Sieck GC. Chronic assessment of diaphragm muscle EMG activity across motor behaviors. Respir Physiol Neurobiol 177: 176 –182, 2011. 25. Mantilla CB, Seven YB, Zhan WZ, Sieck GC. Diaphragm motor unit recruitment in rats. Respir Physiol Neurobiol 173: 101–106, 2010. 26. Mantilla CB, Sieck GC. Phrenic motor unit recruitment during ventilatory and non-ventilatory behaviors. Respir Physiol Neurobiol 179: 57–63, 2011. 27. McPhedran AM, Wuerker RB, Henneman E. Properties of motor units in a homogeneous red muscle (soleus) of the cat. J Neurophysiol 28: 71–84, 1965. 28. Milano S, Grelot L, Bianchi AL, Iscoe S. Discharge patterns of phrenic motoneurons during fictive coughing and vomiting in decerebrate cats. J Appl Physiol 73: 1626 –1636, 1992. 29. Milner-Brown HS, Stein RB, Yemm R. The orderly recruitment of human motor units during voluntary isometric contractions. J Physiol 230: 359 –370, 1973. 30. Prakash YS, Mantilla CB, Zhan WZ, Smithson KG, Sieck GC. Phrenic motoneuron morphology during rapid diaphragm muscle growth. J Appl Physiol 89: 563–572, 2000. 31. Quinn GP, Keough MJ. Experimental Design and Data Analysis for Biologists. Cambridge Univ. Press, 2002. 32. Saboisky JP, Gorman RB, De Troyer A, Gandevia SC, Butler JE. Differential activation among five human inspiratory motoneuron pools during tidal breathing. J Appl Physiol 102: 772–780, 2007.
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measured by the DIAm EMG RMS75. In humans, Butler et al. (8) found no difference in onset discharge rate of diaphragm motor units during voluntary tasks of the same inspiratory flow rate but with different target volumes. However, they found that for voluntary breaths of a constant volume but increased inspiratory flow rate, the initial firing frequencies of DIAm motor units were higher. During the onset of airway occlusion, doublet discharges (instantaneous discharge frequency ⬎ 100 Hz) were sometimes observed, which is also consistent with increased central drive. In paralyzed, mechanically ventilated, and decerebrate cats, doublet discharges of phrenic motor neurons were reported at the onset of fictive vomiting (28). In human limb skeletal muscles, doublet discharges were observed during ballistic contractions (4, 11, 12). Whereas onset discharge rates were comparable during eupnea, hypoxia-hypercapnia, and deep breaths, peak discharge rates were higher during hypoxia-hypercapnia and even higher during deep breaths. Immediately after recruitment, DIAm motor unit discharge rates increased to attain a peak value that varied across motor behaviors. Onset and peak discharge rates of rat DIAm motor units during eupnea and hypoxia-hypercapnia were lower than those reported previously for anesthetized, paralyzed, vagotomized, and mechanically ventilated rats (20). It is likely that removing the inhibitory influence of lung stretch receptors (e.g., by vagotomy) affected inspiratory duration and the peak discharge rates of DIAm motor units. It is also possible that these differences reflect the choice of anesthetic technique. Regardless, examining multiple motor behaviors requiring substantially different levels of force generation permits evaluation of motor unit recruitment order in the same preparation.
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33. Seven YB, Mantilla CB, Zhan WZ, Sieck GC. Non-stationarity and power spectral shifts in EMG activity reflect motor unit recruitment in rat diaphragm muscle. Respir Physiol Neurobiol 185: 400 –409, 2013. 34. Sieck GC, Fournier M. Diaphragm motor unit recruitment during ventilatory and nonventilatory behaviors. J Appl Physiol 66: 2539 –2545, 1989. 35. Sieck GC, Trelease RB, Harper RM. Sleep influences on diaphragmatic motor unit discharge. Exp Neurol 85: 316 –335, 1984. 36. St. John WM, Bartlett D, Jr. Comparison of phrenic motoneuron responses to hypercapnia and isocapnic hypoxia. J Appl Physiol Respir Environ Exercise Physiol 46: 1096 –1102, 1979. 37. Stein RB, Gossen ER, Jones KE. Neuronal variability: noise or part of the signal? Nat Rev Neurosci 6: 389 –397, 2005. 38. Tansey KE, Botterman BR. Activation of type-identified motor units during centrally evoked contractions in the cat medial gastrocnemius muscle. I. Motor-unit recruitment. J Neurophysiol 75: 26 –37, 1996.
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39. Torikai H, Hayashi F, Tanaka K, Chiba T, Fukuda Y, Moriya H. Recruitment order and dendritic morphology of rat phrenic motoneurons. J Comp Neurol 366: 231–243, 1996. 40. Twisk JWR. Applied Longitudinal Data Analysis for Epidemiology: A Practical Guide. Cambridge Univ. Press, 2003. 41. von Euler C, Hayward JN, Marttila I, Wyman RJ. The spinal connections of the inspiratory neurones of the ventrolateral nucleus of the cat’s tractus solitarius. Brain Res 61: 23–33, 1973. 42. Whitelaw WA, Derenne JP, Milic-Emili J. Occlusion pressure as a measure of respiratory center output in conscious man. Respir Physiol Neurobiol 23: 181–199, 1975. 43. Wuerker RB, McPhedran M, Henneman E. Properties of motor units in a heterogeneous pale muscle (m. gastrocnemius) of the cat. J Neurophysiol 28: 85–99, 1965. 44. Zajac FE, Faden JS. Relationship among recruitment order, axonal conduction velocity, and muscle-unit properties of type-identified motor units in cat plantaris muscle. J Neurophysiol 53: 1303–1322, 1985.
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J Appl Physiol 117: 1308–1316, 2014. First published September 25, 2014; doi:10.1152/japplphysiol.01395.2013.
Recruitment of rat diaphragm motor units across motor behaviors with different levels of diaphragm activation Yasin B. Seven,1 Carlos B. Mantilla,1,2 and Gary C. Sieck1,2 1
Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, Minnesota; and 2Department of Anesthesiology, Mayo Clinic, Rochester, Minnesota
Submitted 2 January 2014; accepted in final form 21 September 2014
motor unit recruitment order; recruitment reversal; discharge rates; doublets; electromyography; neuromotor control IN SKELETAL MUSCLES,
increasing levels of force are accomplished by recruiting additional motor units and/or by increasing the discharge frequencies of recruited motor units (1). Based on studying single motor neuron axons in ventral root filaments in spinalized cat, Henneman and colleagues (16, 27, 43) showed that motor units are recruited in a fixed order based on the size-dependent electrophysiological properties (e.g., conduction velocity) of motor neurons. The resulting Henneman Size Principle is now generally accepted and has been demonstrated in numerous skeletal muscles including the diaphragm muscle (DIAm) and across different species including humans (13, 29, 38, 39, 44). Dick and colleagues (13) clearly demonstrated the Size Principle in the cat DIAm but only during hypercapnia in anesthetized and mechanically ventilated animals. Address for reprint requests and other correspondence: G. C. Sieck, Dept. of Physiology and Biomedical Engineering, Mayo Clinic College of Medicine, 200 First St. SW, Rochester, MN 55905 (e-mail: [email protected]). 1308
Several studies have evaluated DIAm motor unit recruitment and discharge rate during eupnea and hypoxia-hypercapnia (7, 8, 14, 32, 35). Generally, these studies found that when ventilation was stimulated by hypoxia and/or hypercapnia, additional motor units were recruited and the peak discharge rates of motor units increased. Butler et al. (8) reported in the human DIAm that motor unit recruitment order across different voluntary tasks was generally fixed with consistent lung volume-related thresholds. However, they reported that reversals of motor unit recruitment order sometimes occurred during voluntary tasks. Voluntary activation of the DIAm is very likely to involve different neuromotor control pathways that may affect recruitment order. Furthermore, although these previous studies examined DIAm motor unit recruitment and discharge frequency during voluntary tasks that accomplished different lung volumes and inspiratory flow rates, they did not directly assess DIAm motor unit recruitment order during motor behaviors of increasing force generation. Compared with eupnea and hypoxia-hypercapnia, greater levels of DIAm force are generated during deep breaths, airway occlusion, and sneezing (25, 34). The goal of this study was to assess the pattern of DIAm motor unit recruitment across motor behaviors that result in increasing force generation. 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. METHODS
Animals. Fifteen adult, male Sprague-Dawley rats weighing 320 – 340 g were used for this study. All experimental procedures and techniques were approved by the Mayo Clinic Institutional Animal Care and Use Committee and were in accordance with the American Physiological Society Animal Care Guidelines. An intramuscular injection of ketamine (90 mg/kg) and xylazine (10 mg/kg) was administered during all experimental procedures. Motor behaviors. Both single motor unit and compound DIAm EMG were recorded during: 1) eupnea (breathing in room air), 2) hypoxia-hypercapnia (breathing in air with 10% O2 and 5% CO2 in N2), 3) deep breath [spontaneous “sighs”; root-mean-squared (RMS) EMG peak ⬎ 2 times eupneic RMS EMG peak value], 4) airway occlusion (sustained closure of airways for ⬃40 s), and 5) sneezing (induced by intranasal administration of capsaicin) as described previously (24, 25, 33). All measurements were made with the animal in supine position with body temperature maintained at 37°C using a heating pad. In analyses of DIAm EMG activity during eupnea and hypoxia-hypercapnia, a 30-s period was used excluding spontaneous deep breaths. During airway occlusion, efforts generated in the last 5-s period of occlusion (4 – 6 efforts) were analyzed. Animals recovered for at least 5 min after each behavior to allow values to return to baseline.
8750-7587/14 Copyright © 2014 the American Physiological Society
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Seven YB, Mantilla CB, Sieck GC. Recruitment of rat diaphragm motor units across motor behaviors with different levels of diaphragm activation. J Appl Physiol 117: 1308 –1316, 2014. First published September 25, 2014; doi:10.1152/japplphysiol.01395.2013.—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% O2 and 5% CO2), deep breaths, sustained airway occlusion, and sneezing. Central drive [estimated by rootmean-squared (RMS) EMG value 75 ms after the onset of EMG activity (RMS75)], 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.
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rates. Double discharges (instantaneous discharge rates ⬎ 100 Hz) were excluded from the analysis of discharge rates. Statistical analysis. Parameters calculated for each burst were averaged across multiple bursts for each motor behavior. The values obtained from the same motor unit across different motor behaviors were treated as repeated measures. Statistics of multiple units were summarized as means ⫾ SE. In the statistical analyses, repeatedmeasures univariate F-test with Greenhouse-Geisser adjustment was used (31, 40). In other comparisons of motor unit discharge properties (e.g., onset and peak motor unit discharge frequencies) across motor behaviors, only those units displaying activity across all behaviors were compared using one-way ANOVA. When F-tests showed significant differences, Tukey-Kramer Honestly Significant Difference test was used to analyze differences post hoc. JMP 9.0 was used to perform statistical calculations (SAS Institute, Cary, NC). Significance level was set to P ⬍ 0.05. RESULTS
Compound DIAm EMG across motor behaviors. Consistent with our previous findings (24, 25, 33), the maximal RMS EMG value (RMSmax) was observed during sneezing (Fig. 1). Thus all RMS values were normalized to RMSmax during sneezing. Representative traces for single RMS EMG bursts across different motor behaviors of the DIAm are shown in Fig. 2A. To estimate neural drive to phrenic motor neurons, the RMS value of the compound DIAm EMG was measured at 75 ms (RMS75) after the onset of activity (Fig. 2B). The RMS75 values during eupnea (15 ⫾ 2%) and hypoxia-hypercapnia (18 ⫾ 2%) were comparable. A slight but significant increase in RMS75 was observed during deep breaths (19 ⫾ 3%; P ⬍ 0.05) compared with eupnea. Importantly, the increase in RMS75 during airway occlusion (42 ⫾ 5%; P ⬍ 0.001) was substantially greater compared with all other motor behaviors. During sneezing, there was a multiphasic pattern, with a relatively low and variable (within and across animals) level of activity at the initiation of a sneeze (for ⬃120 ms) followed by a augmenting increase in RMS DIAm EMG activity that followed a trajectory similar to deep breaths. Finally, there was a third phase with steeper increase in RMS EMG activity. Due
Fig. 1. Representative compound diaphragm EMG recordings and root-mean-squared (RMS) EMG tracings across eupnea, hypoxia-hypercapnia (10% O2, 5% CO2), spontaneous deep breath (sigh), airway occlusion, and sneezing. The main response to hypoxia-hypercapnia was increased respiratory rate (reduced expiratory time). The amplitude of diaphragm EMG activity, as measured by RMS EMG, increased significantly during spontaneous deep breaths (sigh), airway occlusion, and sneezing compared with eupnea and hypoxiahypercapnia.
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Single motor unit and compound DIAm EMG recordings. Single unit and compound DIAm EMG were simultaneously recorded using different electrode and acquisition configurations. To record compound EMG signal, two pairs of teflon-coated, multistranded stainless-steel wires (AS631, Cooner Wire, Chatsworth, CA) were implanted bilaterally into the midcostal DIAm following laparotomy. Insulation was removed for ⬃2 mm at the point of juxtaposition with the DIAm. The compound EMG signal was differentially amplified (2,000⫻), bandpass filtered (20 –1,000 Hz) using an analog amplifier (model 2124, DATA), and digitally sampled at a frequency of 2 kHz using a data-acquisition board (National Instruments, Austin, TX) controlled by a custom-made program (LabView 8.2; National Instruments, Austin, TX). Root-mean squared (RMS) EMG signal was computed from the compound EMG signal using a 50-ms window as described previously (23–25, 33, 34). For single motor unit recordings, each pair of insulated fine-wire electrodes (40 m diameter; Moleculoy, Bridgeport Insulated Wire, Bridgeport, CT) was inserted into the midcostal DIAm. Single motor unit EMG signals were differentially amplified (2,000⫻), bandpass filtered (100 –5,000 Hz) using an analog amplifier (Model EMG100C, Biopac Systems, Goleta, CA), and digitally sampled at a frequency of 20 kHz. Single motor unit action potentials (MUAPs) were identified by a custom thresholding and template-matching algorithm in MATLAB (Mathworks, Natick, MA). Recruitment delay of each motor unit was calculated as the time difference between compound EMG burst onset and the first MUAP of each motor unit within the burst. The average recruitment delay of each motor unit was determined by averaging multiple bursts for each motor behavior. The onset of compound EMG was set as the point where rectified compound EMG value was twice the baseline value. Recruitment order of motor units was determined by analyzing motor unit pairs separately across motor behaviors. The difference in recruitment delays of individual motor units in a pair was evaluated. Recruitment order of a pair of motor units was considered to be reversed if a motor unit in the pair was recruited earlier than the other unit during eupnea but later than the other unit during another motor behavior. The relationships between differences in recruitment delays and the percentage of reversals were also evaluated. Onset and peak discharge rates were calculated as the reciprocal of interspike interval for each motor unit across motor behaviors. Instantaneous discharge rates were filtered with a two-point moving average window to reduce the effects of spontaneous changes in discharge
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to variability at the onset of EMG activity, the RMS75 during sneezing was highly variable and did not reflect the final extent of DIAm EMG activation. Peak RMS EMG activity of the DIAm was 28 ⫾ 3% during eupnea, 37 ⫾ 3% during hypoxia-hypercapnia (P ⬍ 0.01 compared with eupnea), 69 ⫾ 4% during deep breaths (P ⬍ 0.001 compared with eupnea), 62 ⫾ 3% during airway occlusion (P ⬍ 0.001 compared with eupnea), and maximum during sneezing (100%; P ⬍ 0.001 compared with eupnea) (Fig. 2B). The peak RMS DIAm EMG during airway occlusion was comparable to that during deep breaths (P ⫽ 0.89), but peak
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RMS EMG during airway occlusion and deep breaths were significantly greater than hypoxia-hypercapnia (P ⬍ 0.001). DIAm motor unit recruitment across motor behaviors. Figure 3 displays representative compound and single motor unit DIAm EMG activities during a eupneic inspiratory effort. The activities of three motor units were discriminated based on waveform analyses that showed consistent patterns across multiple (⬎10) discharges. In this example, DIAm motor units were recruited in an orderly fashion during the first 100 ms of the eupneic EMG burst duration. Additional motor units contributed to the compound DIAm EMG, but the waveforms of these motor units could not be clearly discriminated across eupneic bursts. After recruitment, motor unit discharge rate progressively increased with peak discharge frequency attained at approximately midway through the effort. In this example, the onset discharge rates were 32, 33, and 29 Hz for motor units 1, 2, and 3, respectively. Peak discharge rates were 48, 47, and 52 Hz, respectively. Most motor units recruited during eupnea could also be discriminated during more forceful DIAm motor behaviors. Motor unit discrimination was more reliable during eupnea and hypoxia-hypercapnia, but during airway occlusion and deep breaths, fewer of those motor units could be discriminated around the peak RMS EMG activity due to amplitude summation/cancellation of additionally recruited motor units. A total of 98 motor units were discriminated across all motor behaviors. Table 1 shows the number of motor units discriminated during each motor behavior and during which behavior those motor units were recruited first. For example, during eupnea 64 motor units were recorded and all of these units were first recruited during eupnea. Of these 64 motor units, the discharge of 55, 48, and 40 of the same motor units could be discriminated during hypoxia-hypercapnia, deep breaths, and airway occlusion, respectively. The number of motor units was decreased, because we were unable to discriminate individual motor units due to amplitude summation/cancellation of multiple motor unit action potentials during more forceful motor behaviors. During hypoxia-hypercapnia, in addition to the 55
Fig. 3. Representative compound DIAm EMG burst and simultaneous single motor unit recordings obtained during eupnea in a spontaneously breathing anesthetized rat. The arrow at top indicates the onset point of compound EMG activity. Three motor units were discriminated via waveform analysis throughout the EMG burst with the superimposed signal shown at left and trains of motor unit action potentials at right for each motor unit. Note differences in time scales between superimposed single motor unit waveforms and compound EMG recordings.
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Fig. 2. Central drive to the diaphragm muscle (DIAm), as measured by RMS DIAm EMG at the initial 75 ms (RMS75), and peak RMS EMG normalized to the maximal RMS EMG value observed during sneezing. A: representative RMS EMG traces averaged within an animal across motor behaviors. Dashed line at 75 ms indicates the point at which RMS75 was calculated (see METHODS for details). B: normalized RMS75 (open bars) and peak RMS EMG (black bars) across DIAm motor behaviors (expressed as % of maximum RMS EMG displayed during sneezing). RMS75 was comparable during eupnea and hypoxia-hypercapnia. Deep breaths presented higher RMS75 value compared with eupnea (P ⬍ 0.05). RMS75 was substantially higher during airway occlusion (P ⬍ 0.001). Peak RMS EMG progressively increased from eupnea to hypoxia-hypercapnia (P ⬍ 0.01) and to deep breath (P ⬍ 0.001). During airway occlusion, peak RMS EMG was comparable to deep breaths (P ⬎ 0.05) but greater than eupnea (P ⬍ 0.001) and hypoxia-hypercapnia (P ⬍ 0.001). *P ⬍ 0.05 vs. eupnea; #P ⬍ 0.05 vs. hypoxia-hypercapnia; &P ⬍ 0.05 vs. deep breaths; §P ⬍ 0.05 vs. RMS75 for same behavior.
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Table 1. Number of motor units recorded across motor behaviors Number of Motor Units Discriminated During Motor Units First Recruited During
Eupnea Hypoxia-hypercapnia Deep breath Airway occlusion Total
Deep breath
Airway occlusion
55 8
48 4 10
40 4
63
62
Eupnea
Hypoxia-hypercapnia
64
64
16 60
Motor units are segregated according to the motor behavior during which they were first recruited.
Fig. 4. Recruitment delays of motor units discriminated across eupnea, hypoxia-hypercapnia, deep breath, and airway occlusion. Motor units were recruited earlier during deep breaths compared with eupnea (P ⬍ 0.01). During airway occlusion, motor units were recruited very early compared with all other motor behaviors (P ⬍ 0.01). *P ⬍ 0.05 vs. eupnea; #P ⬍ 0.05 vs. hypoxiahypercapnia; &P ⬍ 0.05 vs. deep breaths.
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motor units recruited during eupnea, eight new units were discriminated that had not been active previously during eupnea. Of these eight motor units freshly recruited during hypoxia-hypercapnia, the activity of only four units could still be distinguished during deep breaths and airway occlusion. During deep breaths, the activity of 10 additional motor units could be discriminated beyond those that were recruited during eupnea and hypoxia-hypercapnia. During airway occlusion, an additional 16 motor units were freshly recruited. Figure 4 summarizes the recruitment delays of 40 DIAm motor units that were discriminated across all motor behaviors. Recruitment delays were 43.0 ⫾ 5.0 ms during eupnea, 35.0 ⫾ 4.5 ms during hypoxia-hypercapnia, 31.5 ⫾ 4.0 ms during deep breaths, and 14.5 ⫾ 2.0 ms during airway occlusion. These differences in recruitment delays generally corresponded with differences in estimated neural drive to phrenic motor neurons (RMS75; Fig. 2). Compared with eupnea, the recruitment delay during deep breaths was significantly shorter (P ⬍ 0.01). However, the greatest shortening of motor unit recruitment delay was observed during airway occlusion compared with all other motor behaviors (P ⬍ 0.01). Figure 5 shows scatterplots of recruitment delays of the same motor units during eupnea compared with other motor
Fig. 5. Scatterplots displaying recruitment delays of individual motor units during eupnea vs. hypoxia-hypercapnia (A), deep breaths (B), and airway occlusion (C). The identity line is shown in gray. Most motor units were recruited earlier during other motor behaviors compared with eupnea (i.e., lower right of the identity line). Earlier recruitment of motor units was more evident during airway occlusion (shallower slope; m ⫽ 0.37). The r2 values of the fitted lines were 0.47, 0.74, and 0.35 during eupnea vs. hypoxia-hypercapnia, deep breaths, and airway occlusion, respectively. Diaphragm motor units recruited very early (⬍25 ms) during eupnea were recruited both earlier and later during other motor behaviors. However, the motor units with recruitment delays ⬎25 ms during eupnea were almost always recruited earlier during other motor behaviors.
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Fig. 6. Distribution of difference in recruitment delay of motor unit pairs across motor behaviors. The difference in recruitment delay during eupnea is considered as the standard (assumed as positive value) for a motor unit pair and is indicated in the x-axis. An average difference in recruitment delay becoming negative during another motor behavior indicates that a reversal of motor unit recruitment order has occurred. A: motor unit pairs with conserved (i.e., maintained) recruitment order across all motor behaviors. B: motor unit pairs with reversed recruitment order during any motor behavior compared with eupnea. Most recruitment reversals occurred for motor unit pairs with a difference in recruitment delay during eupnea of ⬍20 ms.
DIAm motor unit discharge rates across motor behaviors. Once recruited, the discharge rates of DIAm motor units displayed an augmenting pattern until peak discharge rates were achieved. Figure 7 compares the onset and peak discharge rates of motor units across all motor behaviors. At the onset, the firing frequencies were comparable during eupnea (27 ⫾ 2 Hz), hypoxia-hypercapnia (30 ⫾ 2 Hz), and deep breath (29 ⫾ 2 Hz). However, during airway occlusion, onset firing frequencies were significantly higher compared with all other behaviors (37 ⫾ 3 Hz; P ⬍ 0.01). Higher onset firing frequencies during airway occlusion might be due to increased drive at the onset of the behavior. Occasionally, a short discharge interval (or doublets defined as consecutive MUAPs with instantaneous discharge frequency ⬎100 Hz) was observed at the onset of airway occlusion (Fig. 8). In the example shown
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behaviors. The identity line (shown in gray) indicates the points where a motor unit is recruited with the same delay during eupnea and the other motor behavior being compared. When a motor unit is recruited later during eupnea but earlier during another motor behavior, it is shown in the lower right of the identity line. According to Fig. 5, most of the motor units were recruited earlier during other motor behaviors compared with eupnea, but the extent of deviation from the line of identity varied across motor behaviors. In the graph presented in Fig. 5, slope values lower than unity indicate earlier recruitment of motor units compared with eupnea. Earlier recruitment of motor units was evident across all motor behaviors compared with eupnea (i.e., all slope values ⬍ 1.00). During hypoxia-hypercapnia and deep breaths the slope was 0.64 and 0.72 with r2 values of 0.47 and 0.74, respectively. Earlier recruitment of motor units was most pronounced during airway occlusion as shown by a shallower slope (m ⫽ 0.37, r2 ⫽ 0.35; Fig. 5C). In general, those motor units recruited very early (⬍25 ms) during eupnea were also recruited earlier across other motor behaviors, and in some cases recruitment delays became longer compared with eupnea (above the line of identity; Fig. 5). However, for those motor units with recruitment delays ⬎25 ms during eupnea, they were almost always recruited earlier during other motor behaviors (below the line of identity; Fig. 5). There were eight motor units that were additionally recruited during hypoxia-hypercapnia. These motor units had recruitment delays ranging from 40 to 330 ms (average recruitment delay of 96.7 ⫾ 37.0 ms). During deep breaths, an additional 10 motor units were recruited with recruitment delays ranging from 30 to 550 ms (average recruitment delay of 232.9 ⫾ 41.1 ms). During airway occlusion, an additional 16 motor units were recruited with recruitment delays ranging from 20 to 360 ms (average recruitment delay of 160.2 ⫾ 27.7 ms). Recruitment order of DIAm motor unit pairs across motor behaviors. In addition to single DIAm motor unit analyses, motor units were also analyzed in pairs across more than one motor behavior, and the relative timing of motor unit recruitment could be compared. For example, one motor unit pair was analyzed if two motor units were discriminated. Three motor unit pairs were analyzed for three motor units. Six motor unit pairs were analyzed for four motor units. A total of 104 pairs of motor units were initially discriminated during eupnea and subsequently followed across multiple motor behaviors. Compared with eupnea, the recruitment order of DIAm motor units was conserved for 98%, 87%, and 84% of the motor unit pairs during hypoxia-hypercapnia, deep breath, and airway occlusion, respectively. Figure 6A shows a histogram of differences in recruitment delays between pairs of motor units during eupnea. Although the order of recruitment was generally conserved across motor behaviors, reversals of recruitment order did occur in 2–16% of motor unit pairs depending on motor behavior. In most of the motor unit pairs in which reversal of recruitment order was observed (9 of 13 motor unit pairs), initial differences in recruitment delays during eupnea were ⬍20 ms (Fig. 6B). Among all motor unit pairs, 20% of those with initial differences in recruitment delays during eupnea ⬍20 ms displayed reversal, while for those units with initial differences in recruitment delays during eupnea ⬎20 ms, only 8% showed reversal during another motor behavior.
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DISCUSSION
in Fig. 8, the doublet discharge interval was 9.4 ms (106 Hz) with a sustained discharge rate of 40 –50 Hz. Doublet discharge of motor units was not observed in any other motor behavior in which motor unit discharge could be discriminated. Peak firing frequencies of DIAm motor units were slightly higher during hypoxia-hypercapnia (43 ⫾ 3 Hz) compared with eupnea (37 ⫾ 3 Hz; P ⬍ 0.05, Fig. 7). Peak firing frequencies during deep breaths (61 ⫾ 8 Hz) and airway occlusion (56 ⫾ 7 Hz) were comparable, but higher compared with both eupnea (P ⬍ 0.01) and hypoxia-hypercapnia (P ⬍ 0.01) (Fig. 7).
Fig. 8. Representative single motor unit recordings obtained using fine wire electrodes in the DIAm across motor behaviors (left). Waveform analysis was used to identify the motor unit with the averaged signal and instantaneous discharge frequency (right) shown for this motor unit across behaviors. Notice the doublet discharge (*; discharge frequency ⬎ 100 Hz) at the beginning of airway occlusion.
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Fig. 7. Onset (open bars) and peak (black bars) discharge frequencies across motor behaviors. Onset discharge frequencies of DIAm motor units were greater during airway occlusion compared with eupnea, hypoxia-hypercapnia, and deep breaths (P ⬍ 0.01). Peak discharge rates were significantly higher than onset discharge rates regardless of the motor behavior (P ⬍ 0.01). Peak discharge frequencies of motor units during deep breath and airway occlusion were higher than eupnea and hypoxia-hypercapnia (P ⬍ 0.01). Hypoxiahypercapnia resulted in higher peak discharge frequencies than eupnea (P ⬍ 0.01). *P ⬍ 0.05 vs. eupnea; #P ⬍ 0.05 vs. hypoxia-hypercapnia; &P ⬍ 0.05 vs. deep breaths; §P ⬍ 0.05 vs. RMS75 for same behavior.
The results of the present study support our hypothesis that DIAm motor units are recruited in a fixed order across motor behaviors of varying force, consistent with intrinsic sizerelated electrophysiological properties of phrenic motor neurons (i.e., the Henneman Size Principle). Central drive to phrenic motor neurons (indexed by RMS75 DIAm EMG) was lowest during eupnea and hypoxia/hypercapnia compared with deep breaths and airway occlusion. In particular, central drive was substantially increased during airway occlusion compared with all other motor behaviors. Consistent with this escalation of central drive, single motor unit recruitment delays were shortest during airway occlusion. The motor pattern during sneezing appeared to be multiphasic, with central drive being relatively low and variable during the initial ⬃120 ms, likely reflecting the more complex activation of inspiratory and expiratory muscles in this behavior. Recruitment delays across pairs of simultaneously recorded DIAm motor units were used to determine the order of motor unit recruitment across motor behaviors with different levels of force generation [as indicated by the RMS DIAm EMG (25, 34)]. For these comparisons, motor unit recruitment order was indexed to that observed during eupnea. Accordingly, changes in motor unit recruitment order during different motor behaviors were analyzed only for those motor unit pairs initially recorded during eupnea. However, with increasing central drive in deep breaths and airway occlusion, the number of motor units recruited increased and recruitment delays were shortened. Regardless, compared with eupnea, motor unit recruitment order was mostly maintained (⬎80% of all motor unit pairs across all behaviors). Motor unit pairs displayed reversals of recruitment order primarily when recruitment delays differed by ⬍20 ms in eupnea. Central drive to phrenic motor neurons. The extent of central drive to phrenic motor neurons cannot be directly measured. However, central drive has been estimated by using a number of outcome measures. Previously, we demonstrated that DIAm RMS EMG amplitude is highly correlated with transdiaphragmatic pressure (Pdi; an estimate of DIAm force; r2 ⫽ 0.78) across motor behaviors with different levels of force when the abdomen is bound (25, 34). In the present study, it was not possible to bind the abdomen since fine-wire elec-
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recruitment delays are shortened during motor behaviors associated with more rapid development of force, e.g., increased inspiratory flow rate, airway occlusion, or airway clearance (coughing or vomiting). In a recent study, earlier recruitment of DIAm motor units was confirmed by evaluating the stationarity of DIAm EMG signal, in which changes in EMG mean-square values were assessed by the reverse arrangement test (33). The period of nonstationarity at the onset of the EMG burst (typically ⬍75 ms; hence the selection of the RMS75 measure) correlated well with the period of motor unit recruitment, with both recruitment delays and the period of nonstationarity shortening from eupnea to hypoxia-hypercapnia and to airway occlusion. Motor unit recruitment order is conserved across different force levels. Phrenic motor neurons are thought to receive distributed input from the medulla via premotor neurons (9, 10). Differences in phrenic motoneuron somal and dendritic surface areas (30) likely contribute to orderly recruitment (15, 16, 27, 43) based on their size-dependent electrophysiological properties. Indeed, phrenic motor neurons with slower axonal conduction velocities (smaller motor neurons) are recruited earlier than those with faster conduction velocities (larger motor neurons) (13) in spontaneously-breathing anesthetized cats. Previous studies examining DIAm motor unit activities during different motor behaviors (3, 6, 19, 20, 28, 36) did not compare recruitment order of motor unit pairs in neurally intact animals across motor behaviors requiring substantially different levels of force generation. In the present study, the order of DIAm motor unit recruitment established during eupnea was indeed maintained for 98%, 87%, and 84% of motor unit pairs during hypoxia-hypercapnia, deep breaths, and airway occlusion, respectively. With increasing drive, earlier recruitment of all active motor units is consistent with the slight reduction in the maintenance of motor unit recruitment order. In paralyzed, mechanically ventilated, and decerebrate cats, no reversals were observed in 24 filaments with recordings of motor neuron pairs during fictive coughing, but two of nine motor neuron pairs displayed spontaneous reversals during fictive vomiting (28). It is worth noting that in the present study for cases where DIAm motor unit recruitment order was reversed across behaviors, the difference in recruitment delays during eupnea was very short (20 ms or less). Thus loss of a strict order of motor unit recruitment is consistent with “noise” in neural activation occurring for motor unit pairs with similar electrophysiological properties (37). Taken together, these results are consistent with widely distributed input to phrenic motor neurons and the order of phrenic motor neurons recruitment being based on their size-related electrophysiological properties. Furthermore, these results do not support selective input to phrenic motor neurons emanating from central pattern generators for distinct motor behaviors. In other words, these motor behaviors are distinct in terms of the degree of activation, but not necessarily in other ways. Motor unit discharge rates. All motor behaviors in skeletal muscles are accomplished by controlling both recruitment and discharge frequencies of motor units (1). The onset discharge rates of DIAm motor units were comparable across motor behaviors with the exception of airway occlusion where onset discharge rates were higher. The higher motor unit discharge rate during airway occlusion reflects a greater central drive as
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trodes were dislocated from the DIAm especially during more forceful motor behaviors. Despite the abdomen not being bound in the present study, peak RMS EMG values and the rate of rise of RMS EMG were comparable to those previously reported in rats (25). However, RMS EMG is also influenced by the duration of the inspiratory effort (modulated by lung stretch receptor feedback). Accordingly, DIAm RMS EMG 75 ms after onset (RMS75) was used as a measure of central drive in the present study. Of note, the RMS75 value was very consistent across DIAm EMG bursts within each motor behavior for each animal (coefficient of variation: ⬃10%). The DIAm RMS75 EMG was similar during eupnea and hypoxia-hypercapnia. Previous studies are generally in agreement with a main effect of hypoxia (10% O2)-hypercapnia (5% CO2) on respiratory frequency rather than an increase in peak DIAm EMG activity (and thus tidal volume) in anesthetized rats (24, 25, 33, 34) and paralyzed, mechanically ventilated and decerebrate cats (17). In conscious humans in hyperoxic conditions, exposure to 7% CO2 increased DIAm EMG activity by ⬃20% (slope of the rectified moving average) following a 10-mmHg increase in arterial CO2 (22). Consistently, mouth occlusion pressure at 100 ms after the onset of inspiratory activity (P100) increased from 3 to 7 cmH2O following a 10-mmHg increase in arterial CO2 (2, 21, 42). These discrepancies in the response to hypoxia-hypercapnia may relate to species, anesthetic effects, choice of preparations (decerebrate vs. intact), selection of hypercapnic and/or hypoxic conditions, as well as the method for assessment. Regardless, the change in central drive to phrenic motor neurons during hypoxia-hypercapnia was far from maximal. DIAm motor unit recruitment across motor behaviors of different force levels. Theoretical models of rat DIAm motor unit recruitment suggest that a significant number of DIAm motor units (at least 45–50%) are not recruited during eupnea (25, 26). In general agreement, additional motor units were recruited with increasing central drive compared with eupnea. Unfortunately, limitations related to the discrimination of single motor unit action potentials and volume conduction preclude estimations of the proportion of motor units recruited during each motor behavior and result in fewer motor units being discriminated during more forceful behaviors. The delay in motor unit recruitment for an active motor unit reflects changes in central drive as well as intrinsic, size-related electrophysiological properties of the motor neurons. Phrenic motor neurons receive descending neural drive with time differences on the order of only a few milliseconds (5, 9, 10, 41), at least during eupnea. In the present study, recruitment delays of DIAm motor units varied between 0 and 150 ms during eupnea (43 ms on average). Thus shortened recruitment delays during deep breaths (32 ms) and airway occlusion (15 ms) are consistent with increased central drive during more forceful motor behaviors. These findings are generally consistent with previous studies reporting reduced motor unit recruitment delays in paralyzed, vagotomized, and mechanically ventilated rats (20) and cats (18, 36). These results are also consistent with the observation of Butler et al. (8) for recruitment of DIAm motor units in humans during voluntary tasks. They found that when inspiratory flow increased (perhaps reflecting an increased drive to breathe), motor units were recruited earlier although lung volume threshold remained constant. Overall, these results indicate that DIAm motor unit
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GRANTS This work was supported by National Heart, Lung, and Blood Institute Grant HL-096750 and the Mayo Clinic. DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the author(s). AUTHOR CONTRIBUTIONS Author contributions: Y.B.S., C.B.M., and G.C.S. conception and design of research; Y.B.S. performed experiments; Y.B.S. and G.C.S. analyzed data; Y.B.S., C.B.M., and G.C.S. interpreted results of experiments; Y.B.S., C.B.M., and G.C.S. prepared figures; Y.B.S., C.B.M., and G.C.S. drafted manuscript; Y.B.S., C.B.M., and G.C.S. edited and revised manuscript; Y.B.S., C.B.M., and G.C.S. approved final version of manuscript. REFERENCES 1. Adrian ED, Bronk DW. The discharge of impulses in motor nerve fibres. II. The frequency of discharge in reflex and voluntary contractions. J Physiol 67: i3–151, 1929. 2. Altose MD, Kelsen SG, Stanley NN, Levinson RS, Cherniack NS, Fishman AP. Effects of hypercapnia on mouth pressure during airway occlusion in conscious man. J Appl Physiol 40: 338 –344, 1976. 3. Arita H, Bishop B. Firing profile of diaphragm single motor unit during hypercapnia and airway occlusion. J Appl Physiol 55: 1203–1210, 1983. 4. Bawa P, Calancie B. Repetitive doublets in human flexor carpi radialis muscle. J Physiol 339: 123–132, 1983. 5. Berger AJ. Phrenic motoneurons in the cat: subpopulations and nature of respiratory drive potentials. J Neurophysiol 42: 76 –90, 1979. 6. Bishop B, Settle S, Hirsch J. Single motor unit activity in the diaphragm of cat during pressure breathing. J Appl Physiol 50: 348 –357, 1981. 7. Butler JE. Drive to the human respiratory muscles. Respir Physiol Neurobiol 159: 115–126, 2007.
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8. Butler JE, McKenzie DK, Gandevia SC. Discharge properties and recruitment of human diaphragmatic motor units during voluntary inspiratory tasks. J Physiol 518: 907–920, 1999. 9. Cohen MI, Piercey MF, Gootman PM, Wolotosky P. Synaptic connections between medullary inspiratory neurons and phrenic motoneurons as revealed by cross-correlation. Brain Res 81: 319 –324, 1974. 10. Davies JG, Kirkwood PA, Sears TA. The distribution of monosynaptic connexions from inspiratory bulbospinal neurones to inspiratory motoneurones in the cat. J Physiol 368: 63–87, 1985. 11. Desmedt JE, Godaux E. Ballistic contractions in fast or slow human muscles: discharge patterns of single motor units. J Physiol 285: 185–196, 1978. 12. Desmedt JE, Godaux E. Ballistic contractions in man: characteristic recruitment pattern of single motor units of the tibialis anterior muscle. J Physiol 264: 673–693, 1977. 13. Dick TE, Kong FJ, Berger AJ. Correlation of recruitment order with axonal conduction velocity for supraspinally driven diaphragmatic motor units. J Neurophysiol 57: 245–259, 1987. 14. Gandevia SC, Leeper JB, McKenzie DK, De Troyer A. Discharge frequencies of parasternal intercostal and scalene motor units during breathing in normal and COPD subjects. Am J Respir Crit Care Med 153: 622–628, 1996. 15. Henneman E. Relation between size of neurons and their susceptibility to discharge. Science 126: 1345–1346, 1957. 16. Henneman E, Somjen G, Carpenter DO. Functional significance of cell size in spinal motoneurons. J Neurophysiol 28: 560 –580, 1965. 17. Hwang JC, St. John WM. Facilitation and inhibition of phrenic motoneuronal activities by lung inflation. J Appl Physiol 74: 2485–2492, 1993. 18. Iscoe S, Dankoff J, Migicovsky R, Polosa C. Recruitment and discharge frequency of phrenic motoneurones during inspiration. Respir Physiol Neurobiol 26: 113–128, 1976. 19. Jodkowski JS, Viana F, Dick TE, Berger AJ. Electrical properties of phrenic motoneurons in the cat: correlation with inspiratory drive. J Neurophysiol 58: 105–124, 1987. 20. Kong FJ, Berger AJ. Firing properties and hypercapnic responses of single phrenic motor axons in the rat. J Appl Physiol 61: 1999 –2004, 1986. 21. Lederer DH, Altose MD, Kelsen SG, Cherniack NS. Comparison of occlusion pressure and ventilatory responses. Thorax 32: 212–220, 1977. 22. Lopata M, Evanich MJ, Lourenco RV. Quantification of diaphragmatic EMG response to CO2 rebreathing in humans. J Appl Physiol 43: 262– 270, 1977. 23. Mantilla CB, Greising SM, Zhan WZ, Seven YB, Sieck GC. Prolonged C2 spinal hemisection-induced inactivity reduces diaphragm muscle specific force with modest, selective atrophy of type IIx and/or IIb fibers. J Appl Physiol 114: 380 –386, 2013. 24. Mantilla CB, Seven YB, Hurtado-Palomino JN, Zhan WZ, Sieck GC. Chronic assessment of diaphragm muscle EMG activity across motor behaviors. Respir Physiol Neurobiol 177: 176 –182, 2011. 25. Mantilla CB, Seven YB, Zhan WZ, Sieck GC. Diaphragm motor unit recruitment in rats. Respir Physiol Neurobiol 173: 101–106, 2010. 26. Mantilla CB, Sieck GC. Phrenic motor unit recruitment during ventilatory and non-ventilatory behaviors. Respir Physiol Neurobiol 179: 57–63, 2011. 27. McPhedran AM, Wuerker RB, Henneman E. Properties of motor units in a homogeneous red muscle (soleus) of the cat. J Neurophysiol 28: 71–84, 1965. 28. Milano S, Grelot L, Bianchi AL, Iscoe S. Discharge patterns of phrenic motoneurons during fictive coughing and vomiting in decerebrate cats. J Appl Physiol 73: 1626 –1636, 1992. 29. Milner-Brown HS, Stein RB, Yemm R. The orderly recruitment of human motor units during voluntary isometric contractions. J Physiol 230: 359 –370, 1973. 30. Prakash YS, Mantilla CB, Zhan WZ, Smithson KG, Sieck GC. Phrenic motoneuron morphology during rapid diaphragm muscle growth. J Appl Physiol 89: 563–572, 2000. 31. Quinn GP, Keough MJ. Experimental Design and Data Analysis for Biologists. Cambridge Univ. Press, 2002. 32. Saboisky JP, Gorman RB, De Troyer A, Gandevia SC, Butler JE. Differential activation among five human inspiratory motoneuron pools during tidal breathing. J Appl Physiol 102: 772–780, 2007.
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measured by the DIAm EMG RMS75. In humans, Butler et al. (8) found no difference in onset discharge rate of diaphragm motor units during voluntary tasks of the same inspiratory flow rate but with different target volumes. However, they found that for voluntary breaths of a constant volume but increased inspiratory flow rate, the initial firing frequencies of DIAm motor units were higher. During the onset of airway occlusion, doublet discharges (instantaneous discharge frequency ⬎ 100 Hz) were sometimes observed, which is also consistent with increased central drive. In paralyzed, mechanically ventilated, and decerebrate cats, doublet discharges of phrenic motor neurons were reported at the onset of fictive vomiting (28). In human limb skeletal muscles, doublet discharges were observed during ballistic contractions (4, 11, 12). Whereas onset discharge rates were comparable during eupnea, hypoxia-hypercapnia, and deep breaths, peak discharge rates were higher during hypoxia-hypercapnia and even higher during deep breaths. Immediately after recruitment, DIAm motor unit discharge rates increased to attain a peak value that varied across motor behaviors. Onset and peak discharge rates of rat DIAm motor units during eupnea and hypoxia-hypercapnia were lower than those reported previously for anesthetized, paralyzed, vagotomized, and mechanically ventilated rats (20). It is likely that removing the inhibitory influence of lung stretch receptors (e.g., by vagotomy) affected inspiratory duration and the peak discharge rates of DIAm motor units. It is also possible that these differences reflect the choice of anesthetic technique. Regardless, examining multiple motor behaviors requiring substantially different levels of force generation permits evaluation of motor unit recruitment order in the same preparation.
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33. Seven YB, Mantilla CB, Zhan WZ, Sieck GC. Non-stationarity and power spectral shifts in EMG activity reflect motor unit recruitment in rat diaphragm muscle. Respir Physiol Neurobiol 185: 400 –409, 2013. 34. Sieck GC, Fournier M. Diaphragm motor unit recruitment during ventilatory and nonventilatory behaviors. J Appl Physiol 66: 2539 –2545, 1989. 35. Sieck GC, Trelease RB, Harper RM. Sleep influences on diaphragmatic motor unit discharge. Exp Neurol 85: 316 –335, 1984. 36. St. John WM, Bartlett D, Jr. Comparison of phrenic motoneuron responses to hypercapnia and isocapnic hypoxia. J Appl Physiol Respir Environ Exercise Physiol 46: 1096 –1102, 1979. 37. Stein RB, Gossen ER, Jones KE. Neuronal variability: noise or part of the signal? Nat Rev Neurosci 6: 389 –397, 2005. 38. Tansey KE, Botterman BR. Activation of type-identified motor units during centrally evoked contractions in the cat medial gastrocnemius muscle. I. Motor-unit recruitment. J Neurophysiol 75: 26 –37, 1996.
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39. Torikai H, Hayashi F, Tanaka K, Chiba T, Fukuda Y, Moriya H. Recruitment order and dendritic morphology of rat phrenic motoneurons. J Comp Neurol 366: 231–243, 1996. 40. Twisk JWR. Applied Longitudinal Data Analysis for Epidemiology: A Practical Guide. Cambridge Univ. Press, 2003. 41. von Euler C, Hayward JN, Marttila I, Wyman RJ. The spinal connections of the inspiratory neurones of the ventrolateral nucleus of the cat’s tractus solitarius. Brain Res 61: 23–33, 1973. 42. Whitelaw WA, Derenne JP, Milic-Emili J. Occlusion pressure as a measure of respiratory center output in conscious man. Respir Physiol Neurobiol 23: 181–199, 1975. 43. Wuerker RB, McPhedran M, Henneman E. Properties of motor units in a heterogeneous pale muscle (m. gastrocnemius) of the cat. J Neurophysiol 28: 85–99, 1965. 44. Zajac FE, Faden JS. Relationship among recruitment order, axonal conduction velocity, and muscle-unit properties of type-identified motor units in cat plantaris muscle. J Neurophysiol 53: 1303–1322, 1985.
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