J Appl Physiol 109: 1939 –1949, 2010.
First published October 14, 2010; doi:10.1152/japplphysiol.00812.2010.
Recruitment and rate-coding strategies of the human genioglossus muscle
Julian P. Saboisky,1 Amy S. Jordan,2 Danny J. Eckert,1 David P. White,1 John A. Trinder,2
Christian L. Nicholas,2 Shiva Gautam,1 and Atul Malhotra1
1
Division of Sleep Medicine, Sleep Disorders Program, Brigham and Women’s Hospital and Harvard Medical School,
Boston, Massachusetts; and 2Department of Psychological Sciences, The University of Melbourne, Melbourne,
Victoria, Australia
Submitted 21 July 2010; accepted in final form 9 October 2010
Saboisky JP, Jordan AS, Eckert DJ, White DP, Trinder JA, Nicholas
CL, Gautam S, Malhotra A. Recruitment and rate-coding strategies of the
human genioglossus muscle. J Appl Physiol 109: 1939–1949, 2010. First
published October 14, 2010; doi:10.1152/japplphysiol.00812.2010.—Single
motor unit (SMU) analysis provides a means to examine the motor
control of a muscle. SMUs in the genioglossus show considerable
complexity, with several different firing patterns. Two of the primary
stimuli that contribute to genioglossal activation are carbon dioxide
(CO2) and negative pressure, which act through chemoreceptor and
mechanoreceptor activation, respectively. We sought to determine
how these stimuli affect the behavior of genioglossus SMUs. We
quantified genioglossus SMU discharge activity during periods of
quiet breathing, elevated CO2 (facilitation), and continuous positive
airway pressure (CPAP) administration (inhibition). CPAP was applied in 2-cmH2O increments until 10 cmH2O during hypercapnia.
Five hundred ninety-one periods (each ⬃3 breaths) of genioglossus
SMU data were recorded using wire electrodes(n ⫽ 96 units) from 15
awake, supine subjects. Overall hypercapnic stimulation increased the
discharge rate of genioglossus units (20.9 ⫾ 1.0 vs. 22.7 ⫾ 0.9 Hz).
Inspiratory units were activated ⬃13% earlier in the inspiratory cycle,
and the units fired for a longer duration (80.6 ⫾ 5.1 vs. 105.3 ⫾ 4.2%
inspiratory time; P ⬍ 0.05). Compared with baseline, an additional
32% of distinguishable SMUs within the selective electrode recording
area were recruited with hypercapnia. CPAP led to progressive SMU
inhibition; at ⬃6 cmH2O, there were similar numbers of SMUs active
compared with baseline, with peak frequencies of inspiratory units
close to baseline, despite elevated CO2 levels. At 10 cmH2O, the
number of units was 36% less than baseline. Genioglossus inspiratory
phasic SMUs respond to hypercapnic stimulation with changes in
recruitment and rate coding. The SMUs respond to CPAP with
derecruitment as a homogeneous population, and inspiratory phasic
units show slower discharge rates. Understanding upper airway muscle recruitment/derecruitment may yield therapeutic targets for maintenance of pharyngeal patency.
motoneurons; respiration; tongue; lung; airway; muscle; sleep; apnea
(OSA) affects at least 2– 4% of adults
(24, 54) and has major adverse consequences. Together with
compromised upper airway anatomy, decreased neural drive to
the upper airway muscles during sleep can result in airway
closure and obstructive apnea (e.g., Refs. 2, 35). The genioglossus in humans has been widely studied because it is the
largest upper airway dilator muscle (1, 2, 12, 49), is readily
accessible (3, 21, 44), and its activity is thought to be representative of phasic upper airway dilator muscles. While pharmacological agents that increase genioglossal muscle activity
(such as ampakines) may be a viable strategy for OSA therapy
OBSTRUCTIVE SLEEP APNEA
Address for reprint requests and other correspondence: J. Saboisky, Division
of Sleep Medicine, Sleep Disorders Program, Brigham and Women’s Hospital,
221 Longwood Ave., Boston, MA 02115, United States of America (e-mail:
JSABOISKY@partners.org).
http://www.jap.org
(e.g., Refs. 28, 41, 53), the ideal therapeutic targets remain
unclear.
Given that genioglossus muscle activation appears necessary
and sufficient to stabilize breathing in OSA patients during
sleep (23, 25), understanding the responses to both chemoreceptive and mechanoreceptive stimuli is critical to upper airway motor control. Chemoreceptor activation (increased CO2)
increases hypoglossal motor output, as indicated by multiunit
electromyographic (EMG) activity of respiratory muscles of
animals and humans (7, 22, 27, 31). Continuous positive
airway pressure (CPAP) decreases the “phasic” multiunit EMG
signal (4), and the signal can be completely abolished after
anesthetizing the upper airway (13). CPAP raises the transmural airway pressure (i.e., reduces negative airway pressure) and
increases lung volume to prevent airway collapse (4). However, the effect of CPAP on genioglossal single motor units
(SMUs) has not been investigated.
As motoneurons are the final common pathway of neural
output, assessing their responses to stimuli provides insight
into motor control of a muscle. The regulation of skeletal
muscle force/output is controlled in two interactive ways:
1) changing the number of units that are active at any moment
with recruitment/derecruitment, and 2) by varying the discharge frequency and pattern of action potentials of SMUs
(rate coding) (for review, see Ref. 10). During eupneic breathing, SMU activity of the human genioglossus exhibits discharge patterns that both correlate to the respiratory cycle
(inspiratory phasic, inspiratory tonic, expiratory phasic, and
expiratory tonic units), while other units have no distinct
respiratory modulation (tonic and tonic other units) (38). However, the relative contribution of recruitment and rate coding
over the physiological range is largely unknown for the upper
airway. Two recent studies have demonstrated increased recruitment of SMUs with mild hypercapnic stimulation in
healthy young populations without increases in rate coding (30,
36). Similarly, at arousal from sleep, the increased EMG
activity is also primarily due to recruitment, with a predominant increase in the number of inspiratory phasic units, and not
through rapid increases in peak discharge rates (51). However,
while these studies have demonstrated recruitment of previously silent units, the units that are already active may be
capable of augmenting their discharge rate (possibly in a
narrower range), in response to increased total muscle output
(8). Consistent with the above studies (30, 36, 51), at the
alpha-to-theta transition, there was primarily derecruitment of
previously active inspiratory units, and those that continued to
discharge reduced their duty cycle (52). However, the tonic and
expiratory classes of units seem to be unaffected at sleep onset,
showing an absence of derecruitment and no alteration in their
discharge pattern (52).
8750-7587/10 Copyright © 2010 the American Physiological Society
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GENIOGLOSSUS BEHAVIOR WITH INCREASED VENTILATION AND CPAP
Therefore, we aimed to determine how the different discharge patterns modify their frequency or pattern of discharge
to chemoreceptive and mechanoreceptive stimuli. We hypothesized that the different classes of motor units, previously
described (38), would respond as a single population through
increases in rate coding (increased firing frequency) and earlier
activation of active units, together with recruitment of additional inspiratory phasic units under higher conditions of elevated CO2 (10). In addition, because premotor inputs and their
various thresholds for activation may alter the activation patterns (6, 22), we hypothesized a marked reduction in the firing
rates and derecruitment of inspiratory units with CPAP. Some
of the results of these studies have been previously reported in
the form of abstracts (37, 42). Given the importance of these
muscles in stabilizing breathing, insights into recruitment and
rate-coding strategies of genioglossal activity could have therapeutic implications for sleep apnea.
METHODS
Complete data were successfully obtained in 15 healthy human
subjects. Demographic details of the group are given in Table 1.
Twenty-one subjects were studied in total; however, six subjects were
excluded due to trouble with vigilance (n ⫽ 2), the observation of
erratic breathing patterns (n ⫽ 1), end-tidal CO2 (PETCO2) unstable
(n ⫽ 1), or no decomposable SMU activity (n ⫽ 2). The data for the
excluded subjects are not included in Table 1. All subjects gave
written, informed consent before participation in this study, which
was approved by the local Human Research Committee of the
Brigham and Women’s Hospital and conformed to the Declaration of
Helsinki.
General procedures. To determine the depth and location for the
recording electrodes in genioglossus, the local anatomy of the upper
airway musculature was examined with ultrasonography (12L highfrequency linear array transducer, Vivid i GE Healthcare, Chalfont St.
Giles, Bucks, UK) (14, 38, 39). The distance from the skin to the
inferior margin of the genioglossus and geniohyoid muscles and the
lateral width of genioglossus were recorded using an electronic caliper
(14). Before the insertion of wires, a topical anesthetic cream was
placed under the chin surface for a minimum of ⬃30 min. The chin
was thoroughly cleaned using disposable topical antiseptic wipes, and
a small reference cross was drawn under the chin 10 mm posterior to
genial turbercle, using a sterile skin marker (DEVON tyco/Healthcare
Group, Chicopee, MA). Since this area had been examined by ultrasonography, we defined in each individual subject the minimal depth
to obtain recordings from the correct muscle and marked this on the
Table 1. Subject demographics
Group Subject Characteristics
Subjects, no.
Female/male, no.
Age, yr
Height, cm
Weight, kg
Body mass index, kg/m2
Units per subject, no. (range)
Inferior margin of geniohyoid muscle, mm
Inferior margin of genioglossus muscle, mm
Genioglossus width, mm
15
5/10
32.9 ⫾ 3.1
172.7 ⫾ 1.9
68.8 ⫾ 4.0
23.8 ⫾ 0.7
6.3 ⫾ 1.3 (1–19)
12.4 ⫾ 0.9
20.7 ⫾ 1.0
15.4 ⫾ 0.5
Values are means ⫾ SE, unless otherwise specified. Shown are the number
of subjects, female-to-male ratio, age, height, weight, body mass index,
number of units, and ultrasound measurements of the upper airway. Ultrasound
measurements from the skin to the inferior margin of the geniohyoid and
genioglossus muscles and the mean width genioglossus are also given.
J Appl Physiol • VOL
needle (see Table 1). The three 27-gauge needles were inserted at 90°
to the skin surface and advanced through the mylohyoid and geniohyoid muscles into the genioglossus (⬎21 mm) with the subjects
relaxed, lying comfortably supine. The lumen of each hypodermic
needle contained a single Teflon-coated 51-␮m hooked stainless steel
fine-wire electrode (A & M Systems, Carlsborg, WA) with ⬃0.5-mm
recording area exposed. The three fine-wire electrodes were inserted
under the chin; the most anterior electrode was positioned a minimum
of 10 mm posterior to the inner border of the mental protuberance and
just lateral to the midline; and the two posterior electrodes were
positioned a further ⬃10 mm behind the anterior electrode ⬃5 mm
lateral to each side of the midline. The fine-wire electrodes were
referenced to a surface electrode positioned over the bony mandible
(MEDI-TRACE 100 series, Kendall Healthcare, Mansfield, MA). A
large flexible ground electrode (1180, 3M Health Care, St. Paul, MN)
was placed over the right clavicle. Genioglossus EMG signals were
filtered (30 Hz to 3 kHz), amplified (⫻1,000 –10,000) (Grass Instruments), sampled at 10 kHz, and stored on computer for offline
analysis (Spike2 with 1401 interface, Cambridge Electronic Design,
Cambridge, UK). Genioglossus EMG activity was recorded over the
entire protocol (see below).
For the entire protocol, the subjects breathed through a nasal mask
(Gel Mask, Respironics, Murrysville, PA) attached to a heated pneumotachograph (model 3700A, Hans-Rudolph, Kansas City, MO) and
a differential pressure transducer (Validyne, Northbridge, CA). Airway pressure and PETCO2 at the nares were measured through ports in
the nasal mask. The flow signal was integrated to give volume. To
increase and hold the PETCO2 constant, the pneumotachograph was
connected in series to a bleed port for compressed medical air (dry air:
79% N2, 21% O2) titration, and two corrugated respiratory tubes (each
25 mm in diameter ⫻ 180 cm length) were connected directly to a
CPAP device (Philips Respironics, Murrysville, PA).
Throughout the procedure, subjects lay supine comfortably and
relaxed, breathed quietly, and remained awake. Wakefulness was
confirmed either with the investigators directly watching the subjects
or by monitoring the electroencephalogram (C3-A1, Oz-A2; based on
␣- or ␤-EEG activity).
Protocol. The subjects were asked to lie comfortably supine while
remaining awake during the following protocol.
1) A baseline period of 3 min of data collection enabled a stable
recording with the PETCO2 levels monitored and recorded.
2) Two corrugated respiratory tubes to increase the dead space
were connected to the mask, and, for ⬃3 min, compressed medical air
was turned on to a level at which the PETCO2 was maintained at similar
levels to the baseline flow rate.
3) In rebreathing, the flow rate of the compressed medical air was
then reduced to a point at which rebreathing ensued (⬃60 s) until peak
PETCO2 elevation was 10 Torr. The EMG signal was also used to
regulate the peak PETCO2, as when SMU activity approached near
saturation for decomposition, the PETCO2 level was maintained.
4) CPAP was then applied at 2-cmH2O increments for 1 min until
a level of 10 cmH2O was obtained. Hypercapnia through rebreathing
was maintained during CPAP application.
5) CPAP was terminated.
Sorting of SMU activity. Motor unit potentials were extracted from
the raw EMG signal offline using a spike-triggered threshold. The
individual SMUs were sorted into “templates” based on their detailed
morphology and amplitude (Spike2 analysis system, Cambridge Electronic Design). Manual inspection of all motor unit potentials confirmed the sorting. Instantaneous frequency plots were derived from
the time of discharge of the unit (Fig. 1).
The activity of each motor unit was then classified based on its
pattern of discharge during the respiratory cycle, which was determined from the airflow signal (see Fig. 4) (38, 39). Briefly, all units
were classified into tonic or phasic categories, depending on whether
they discharged throughout both inspiration and expiration (tonic), or
only during either inspiration or expiration (phasic). Units were
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GENIOGLOSSUS BEHAVIOR WITH INCREASED VENTILATION AND CPAP
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Fig. 1. Typical example of two concurrently recorded single motor units during quiet breathing to show the measurements used in the analysis. From bottom
to top: raw electromyographic (EMG; at left) with overlaid motor unit potentials from units (at right of the instantaneous frequency plots), the corresponding
volume signal, and the instantaneous frequency plot of the two units (dashed line represents the running average of the unit calculated over 200 ms). The shaded
area represents the inspiratory time (TI) for which the discharge times were all normalized (0% TI and 100% TI). The vertical dashed lines represent the TI (0%
TI and 75% TI) for which calculations were performed on the percentage of the number of units active or not active. The various measurements are shown for
both the top inspiratory tonic unit and the lower inspiratory phasic unit, including the onset time (dti) from the first motor unit potential in each breath (or where
the frequency first increased for the tonic unit) and onset frequency (fi) taken from the first interspike interval in each breath, and the end time (dte) and end
frequency (fe) taken from the last potential in each breath (or where the frequency decreased for the tonic unit). The time of the peak frequency (dtp and fp)
was measured from a running average over 200 ms (dashed line). For each unit, these variables were measured for three breaths and then averaged.
further classified according to the timing of their peak activity:
“inspiratory phasic” motor units discharged phasically with their peak
frequency during inspiration (see Fig. 2); “expiratory phasic” units
discharged phasically with their peak frequency during expiration;
“inspiratory tonic” units discharged through inspiration and expiration, but increased their discharge frequency during inspiration; and
“expiratory tonic” units discharged through inspiration and expiration,
but increased their discharge during expiration (see Fig. 3). “Tonic”
units discharged throughout inspiration and expiration with no obvious respiratory modulation of discharge frequency. A small number of
units discharged continuously with some modulation, but not in time
with the respiratory cycle, and they were classified as “tonic other”
units. In addition to the visual categorization of the different classes of
motor unit activity, cross correlations between volume (from the
integrated flow signal) and instantaneous firing frequency (smoothed
over 200 ms) were calculated for all possible phase differences
between the two signals on a breath-by-breath basis (see Supplemental Table S1; the online version of this article contains supplemental
data). The strength of each correlation was evaluated by calculating
the linear coefficient of determination (r2) and the timing of the
maximal value of this coefficient. The respiratory phase of the
maximal r2 (lag time) indicated whether the unit had a predominant
inspiratory or expiratory modulation. Figure 4 displays the confirmation of the computed cross correlations with a plot of the r2 value
against the lag time (s) for all units in the eight conditions (A–H).
Measurement of respiratory variables and discharge properties of
SMUs. Respiratory variables, including inspiratory time (TI; s), expiratory time (s), minute ventilation (l/min), respiratory frequency, tidal
volume (liter), and PETCO2 (Torr) were analyzed over the breaths,
where the motor units were sorted with the aid of custom-designed
semi-automated software (Spike2, Cambridge Electronic Design);
averaged means were calculated for each of the conditions.
Measurements of SMU discharge behavior were derived from
instantaneous frequency plots for each motor unit (Spike2 Cambridge
Electronic Design, script courtesy of the Gandevia Laboratory, Randwick, Sydney, Australia). Discharge timing was expressed relative to
the integrated flow signal. Motor units were measured over three
J Appl Physiol • VOL
continuous breaths in each category or until the unit became inactive
(baseline, CO2, 2 cmH2O ⫹ CO2, 4 cmH2O ⫹ CO2, 6 cmH2O ⫹ CO2,
8 cmH2O ⫹ CO2, 10 cmH2O ⫹ CO2, and with CPAP off).
However, in rare circumstances, units were accepted if sorted for
two consistent breaths. Contamination of the EMG signal due to
events such as sighs or swallows was avoided in this analysis by
discarding these data.
For phasic units, the onset discharge time was measured at the first
discharge for each breath, and the end time was measured at the last
discharge in each breath (See Fig. 1). For inspiratory and expiratory
tonic units, onset time was taken visually from when the discharge
frequency modulation first increased above the tonic levels, and the
end time when the discharge frequency first returned to the tonic level
(See Fig. 1). Onset discharge frequency for phasic units was calculated from the first interspike interval for each breath. For inspiratory
tonic and expiratory tonic units, onset firing frequency was measured
at the first increase in the discharge frequency above the tonic level.
Peak discharge frequencies were derived from the peak of the instantaneous frequency with a running average for each breath (smoothed
over 200 ms).
All variables were averaged across the sorted consecutive breaths.
Statistical differences for the discharge parameters were assessed
using one-way (ANOVA) with Student-Newman-Keuls post hoc
analysis. If data were not normally distributed, then the KruskalWallis test was applied or the two-way ANOVA was performed on
ranks (SigmaPlot 11). Statistical significance was defined by P ⬍
0.05. Units that met the criterion for the inspiratory phasic and
inspiratory tonic classes under the conditions, CO2, 4 cmH2O, 6
cmH2O, 8 cmH2O, and 10 cmH2O (unit must have been active and not
changing class), were selected for a one-way repeated-measures
analysis using Student-Newman-Keuls post hoc analysis; one-way
repeated-measures ANOVA on ranks was used where units were not
normally distributed. The population analysis was calculated using a
mixed-model analysis, taking into consideration the subject, number
of units, class of the units, condition, and peak firing rates (SAS
statistics). Values are given as means ⫾ SE.
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GENIOGLOSSUS BEHAVIOR WITH INCREASED VENTILATION AND CPAP
Fig. 2. Recording of single motor unit activity in the genioglossus. A typical example of a recording from a single subject, in each of the eight conditions [baseline,
CO2, 2 cmH2O, 4 cmH2O, 6 cmH2O, 8 cmH2O, 10 cmH2O, and continuous positive airway pressure (CPAP) off]. Traces from bottom to top: mask pressure,
end-tidal CO2 (PETCO2), volume, raw EMG from two electrode sites (a and b), with both rectified and integrated EMG (time constant, 100 ms, calibration 0 –10
␮V), instantaneous frequency plots for three units, and the superimposed motor unit potentials for each unit (inset). The simultaneously recorded units increased
their discharge in phase with inspiration. All three units were classed as inspiratory phasic (see Fig. 1 and METHODS for description of the classification). With
CO2, the units tended to increase their activity, and graded suppression was observed with the application of positive airway pressure. Unit 1 was recorded on
EMG (a), and units 2 and 3 were recorded on EMG (b). Inset calibrations: 100 ␮V and 2 ms.
RESULTS
Successful unitary recordings, using selective intramuscular
fine-wire electrodes, were made from 96 SMUs in the genioglossus muscle during normal eupnic breathing, hypercapnic
breathing (through a rebreathing circuit), and under five levels
of CPAP. A total of 591 sections (each ⬃3 breaths) of genioglossus SMU data were sorted and analyzed in this study.
Typical recordings for each of the eight conditions are shown
in Figs. 2 and 3. Concurrent recordings of multiple SMUs were
obtained in Fig. 2. In this example, units 1 and 2 were both
suppressed by the application of CPAP, and, to confirm the unit
was not “lost” due to displacement of the electrode, the units
were also decomposed in the CPAP off condition. Figure 3
illustrates the activity of an expiratory tonic motor unit that was
not suppressed with the application of CPAP through the eight
experimental conditions. This unit displays multiple peak discharges that may be explained by convergence of two pacemakers (see Fig. 3B). The correlation and lag time calculated
between each unit’s firing frequency (smoothed over 200 ms)
with the signal of lung volume, for each of the six classes of
units in the genioglossus muscle, in each of the eight conditions are shown in Fig. 4, A–H. Figure 4 shows the timing of
the peak correlation [time (s)] for each motor unit plotted
against strength of the coefficient of determination (r2). Inspiratory units had a peak r2 before the end of inspiration
(negative times), and expiratory units had a peak r2 after the
end of inspiration during expiration (positive times; see METHJ Appl Physiol • VOL
ODS and Refs. 38 – 40). In each of the eight conditions, the
activity of 42–94 SMUs was recorded, and the predominant
class of motor unit activity exhibited was inspiratory phasic
activity (Fig. 5, see Table 3; P ⬍ 0.01, mixed-model analysis).
Ventilatory data. Respiratory parameters were altered with
the application of CPAP and the rebreathing protocol (see
Table 2). There were no significant differences for TI, expiratory time, and respiratory frequency between the eight conditions. Values for minute ventilation tended to be elevated under
all conditions compared with baseline and were significantly
greater than baseline at the higher levels of CPAP 4 –10
cmH2O (P ⬍ 0.05). Similarly, tidal volume increased under all
levels of CPAP compared with baseline (P ⬍ 0.05). PETCO2
was elevated under the CO2 condition by 6 ⫾ 0.3 Torr and
remained 12% higher until the CPAP off condition (P ⬍ 0.05).
Changes in the number of units and discharge characteristics with rebreathing. Compared with the baseline condition,
significantly more (32%) SMUs were recorded under the condition of elevated CO2 (63 vs. 93; P ⬍ 0.01, mixed-model
analysis). During the hypercapnic condition, the inspiratory
phasic units increased their peak discharge by ⬃2 Hz (17.9 ⫾
0.8 vs. 20.1 ⫾ 0.7 Hz; P ⫽ 0.037; see Fig. 6). In addition, the
inspiratory phasic motor units were recruited earlier in inspiration under the CO2 condition by an average of ⬃16% TI
(%TI; see Fig. 1 for how the timing variables are defined, P ⫽
0.02; ␹2). There were no such differences for the time to peak
firing and end-firing time for inspiratory phasic units, which
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GENIOGLOSSUS BEHAVIOR WITH INCREASED VENTILATION AND CPAP
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Fig. 3. Genioglossus single motor unit activity during the eight experimental conditions. A: representative example of a recording from a single subject through
the eight conditions. Traces from bottom to top, the mask pressure through (left to right), PETCO2, volume, the raw EMG from one electrode site, rectified and
integrated EMG (time constant 100 ms, calibration 0 –10 ␮V), instantaneous frequency plots for a single unit, and the superimposed motor unit potentials for
the unit under each condition. Calibrations: 250 ␮V and 2 ms. B: expanded section of 2 and 6 cmH2O. Note that, in the 2-cmH2O condition, the unit clearly
increases its discharge in phase with expiration and was classed as expiratory tonic 0.80 r2. However, at 6 cmH2O, this unit displayed multiple peaks, both
inspiratory and expiratory, while it maintained a reduced expiratory peak cross-correlation with the volume signal 0.59 r2 (see METHODS for description of the
classification).
occurred at the same time under baseline and CO2 conditions
(peak times 39.8 ⫾ 3.8 vs. 44.2 ⫾ 3.5% TI; end times 81.8 ⫾
6.2 vs. 95.9 ⫾ 3.7% TI, P ⬎ 0.05). Despite no significant
change in end-firing times, the number of units that continued
to fire for longer than 75% TI was significantly increased from
baseline to the CO2 condition (20 vs. 80%, P ⬍ 0.001; ␹2). The
increase in the number of units firing over 75% TI was also
seen in the inspiratory phasic units as 43% stopped discharging
in the baseline condition; only 20% of units ceased discharge
in the CO2 condition (P ⬍ 0.001, ␹2).
In contrast, the population of inspiratory tonic SMUs decreased their peak discharge frequency from 25.6 ⫾ 1.3 to 22.2 ⫾
1.0 Hz from the baseline to CO2 condition, but Fig. 6 illustrates
this was largely due to the newly recruited units discharging at
lower peak frequencies (Kruskal-Wallis Dunns post hoc; P ⬍
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0.05). While it appeared that an additional 10% of inspiratory
tonic motor units commenced before the onset of inspiratory
flow, this was not significant (P ⫽ 0.15; ␹2). All of the
inspiratory tonic units discharged at over 75% TI at an elevated
rate (above the expiratory tonic frequency) during the baseline
condition. During the CO2 condition, there was a little reduction (⫺10%) in the number of units that maintained this firing
at an elevated rate of over 75% TI.
Changes in the number of units and discharge characteristics with positive airway pressure. While there was no change
in the discharge rates of individual motor units with CPAP,
there was an abrupt and marked change in the numbers of units
recorded. Once CPAP reached 6 cmH2O, there were fewer
units active than in the increased CO2 condition (67 units,
CPAP ⫹ CO2 vs. 93 units CO2 alone; P ⬍ 0.01, mixed-model
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GENIOGLOSSUS BEHAVIOR WITH INCREASED VENTILATION AND CPAP
Fig. 4. Classification of single motor unit in genioglossus based on their firing frequency-volume coefficient of determination. A–H: plots of the strength
(coefficient, r2) and the time of the peak cross correlations for each unit (mean). The correlations were calculated between the volume signal and the discharge
frequency (smoothed over 200 ms) of the motor unit by computing all possible x-axis phase differences between the two signals. Time zero represents the end
of inspiration. Units were classified into inspiratory phasic (solid circles), inspiratory tonic (half-solid circles), expiratory phasic (solid squares), expiratory tonic
(half-solid squares), and tonic and tonic other units (plus signs). Inspiratory units had a peak r2 before the end of inspiration (negative times), and expiratory units
had a peak r2 after the end of inspiration during expiration (positive times). Inspiratory phasic r2 values for CO2 condition were greater than 10-cmH2O condition
(P ⬍ 0.05). The inspiratory tonic units had a trend toward alterations in the lag times of these units between the baseline (⫺1.2 ⫾ 0.1 s), 8 cmH2O (⫺0.9 ⫾
0.1 s), and 10 cmH2O (⫺0.8 ⫾ 0.1 s; all nonsignificant).
analysis; see Table 3, Fig. 5, and Fig. 7). This suppression
effect was more marked as the pressure increased to 10
cmH2O, when there were fewer units active than under the
baseline condition (42 CPAP ⫹ CO2 vs. 63 baseline units; P ⫽
0.01 mixed-model analysis).
There was a significant change in the timing of motor unit
activity over the conditions of increased CPAP levels. The
number of inspiratory phasic units with recruitment times
before inspiratory flow commenced were initially 31% with
CO2, and the number declined to 21% during the period of 10
cmH2O (P ⬍ 0.001, ␹2 test). The inspiratory phasic units with
end-firing times before 75% TI progressively increased from
20% under the CO2 condition to a maximum of 47% at 10
cmH2O (P ⬍ 0.001). The graded reduction reached significance at 6 cmH2O with 36% of units ending their firing (P ⫽
0.02). The number of units that were active after 75% TI
returned to control values with CPAP off.
The background “tonic” discharge frequency of the inspiratory tonic units was similar between conditions (baseline: 14.0 ⫾
0.9 Hz; CO2: 12.0 ⫾ 0.6 Hz; 2 cmH2O: 11.8 ⫾ 0.9 Hz; 4
cmH2O: 16.0 ⫾ 4.0 Hz; 6 cmH2O: 10.8 ⫾ 0.8 Hz; 8 cmH2O:
12.0 ⫾ 0.7 Hz; 10 cmH2O: 9.7 ⫾ 0.8 Hz; CPAP off: 13.1 ⫾
0.8 Hz, P ⫽ 0.057). Due to the low number of units in the
expiratory (tonic and phasic) and tonic classes (see Table 3),
detailed subanalyses were not powered to detect changes.
J Appl Physiol • VOL
Repeated-measures analysis for inspiratory phasic motor
units during CO2 and CPAP conditions. In these experiments,
we attempted to follow motor units throughout the entire protocol.
However, with the recruitment and derecruitment of motor units
under the eight conditions, a subanalysis with repeated measures
could be performed on only 20 inspiratory phasic motor units.
These units were chosen as they remained active through the
conditions CO2, 4 cmH2O, 6 cmH2O, and 8 cmH2O. Thus the
analysis was performed to examine the progressive effects of
increased CPAP on the mechanoreceptor activation.
There were a reduced number of motor unit action potentials
within each breath under CPAP compared with the CO2 condition. Under the CO2 condition, the mean number of motor unit
action potentials was 34.4 ⫾ 3.9, and this declined to 24.1 ⫾ 3.0
at 4 cmH2O, 20.4 ⫾ 3.5 at 6 cmH2O, and 21.0 ⫾ 4.6 at 8 cmH2O
(see Fig. 2 for raw data; P ⬍ 0.05). Despite no change in the onset
discharge rates, the peak discharge frequency for the 20 inspiratory phasic SMUs was found to have significant reductions at
8-cmH2O CPAP (18.4 ⫾ 1.2 Hz) compared with CO2 (21.7 ⫾ 1.1
Hz; P ⫽ 0.03).
The timing of the motor units relative to the onset of flow
was also altered. The preinspiratory firing during CO2 (⫺3.7 ⫾
6.3% TI) was delayed at 4 cmH2O (6.9 ⫾ 4.3% TI), 6 cmH2O
(12.9 ⫾ 5.7% TI), and 8 cmH2O (7.7 ⫾ 5.0% TI; all P ⬍ 0.05).
Consistent with fewer motor unit potentials activated, the
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GENIOGLOSSUS BEHAVIOR WITH INCREASED VENTILATION AND CPAP
Fig. 5. The time and frequency plots. A–H: each individual unit in each condition. Each horizontal line represents the firing of a genioglossus single motor unit
(591 in total). Vertical dotted lines represent the onset (0%) and end (100%) of inspiration. For each motor unit, the onset time (small colored circle on left) of
firing is joined by a thick horizontal line to its end discharge time (small colored circle on right). The color of the circles indicates the onset and end firing
frequencies (see inset). The mean peak firing frequency is indicated by the color of the thick horizontal line, and the time of the peak firing frequency is indicated
by a black circle. For units that discharged throughout both phases of the respiratory cycle, a thin horizontal line is colored to code the tonic firing frequency.
Units are ordered in each category (phasic or tonic), according to their onset discharge time. With hypercapnic stimulation, the peak discharge rate for the
population is increased, and more single motor units were activated (63 vs. 93; both P ⬍ 0.01). A greater percentage of the inspiratory phasic motor units was
activated before the onset of flow 0% TI (20%, P ⫽ 0.02) and continued to discharge after 75% TI (60%, P ⬍ 0.001). The stepwise application of CPAP led
to reduced discharge frequencies for the population at 6, 8, and 10 cmH2O (P ⬍ 0.01). The firing times were also markedly altered under CPAP with a 22%
TI decrease in the duration of discharge at 8 cmH2O. The onset firing times of these units that were preinspiratory with CO2 (⫺3.7% TI) were delayed with the
application of 8-cmH2O CPAP to postinspiratory activation (7.7% TI).
end-firing times also showed a consistent and progressive
reduction from the CO2 to the 8-cmH2O condition, with the
end time reduced from 94.8 ⫾ 5.0 to 77.8 ⫾ 6.4% TI (see Fig.
5 for the motor unit responses; P ⬍ 0.05).
DISCUSSION
This study provides new data on the adjustments in human
genioglossus motor unit discharge behavior in response to
Table 2. Ventilatory data during electromyographic recordings, one value for each subject/condition
Inspiratory time, s
Expiratory time, s
Minute ventilation, l/min
Respiratory frequency
Tidal volume, liter
End-tidal CO2, Torr
Baseline
CO2
2 cmH2O
4 cmH2O
6 cmH2O
8 cmH2O
10 cmH2O
CPAP Off
1.9 ⫾ 0.2
2.7 ⫾ 0.2
7.7 ⫾ 0.7
14.4 ⫾ 0.9
0.6 ⫾ 0.1
39.7 ⫾ 0.8
2.0 ⫾ 0.1
2.3 ⫾ 0.1
13.8 ⫾ 1.0
14.8 ⫾ 0.8
1.0 ⫾ 0.1
46.1 ⫾ 0.6*†
1.9 ⫾ 0.1
2.5 ⫾ 0.2
15.9 ⫾ 1.1*
14.6 ⫾ 0.8
1.1 ⫾ 0.1*
46.2 ⫾ 0.6*†
1.8 ⫾ 0.1
2.7 ⫾ 0.3
15.9 ⫾ 1.2*
14.7 ⫾ 0.9
1.1 ⫾ 0.1*
45.9 ⫾ 0.6*†
1.8 ⫾ 0.1
2.8 ⫾ 0.3
17.0 ⫾ 1.3*
15.2 ⫾ 1.0
1.2 ⫾ 0.1*
45.8 ⫾ 0.5*†
1.8 ⫾ 0.1
2.8 ⫾ 0.3
17.5 ⫾ 1.2*
14.9 ⫾ 1.0
1.2 ⫾ 0.1*
45.2 ⫾ 0.7*†
1.8 ⫾ 0.2
2.8 ⫾ 0.3
16.4 ⫾ 1.4*
14.9 ⫾ 1.5
1.2 ⫾ 0.1*
44.5 ⫾ 0.8*†
2.0 ⫾ 0.2
2.4 ⫾ 0.3
14.0 ⫾ 1.3
15.5 ⫾ 1.0
1.0 ⫾ 0.1
41.4 ⫾ 0.7
Values are means ⫾ SE. For each variable, a mean value was derived from the mean for each subject under each condition (N ⫽ 15). There were no significant
differences for inspiratory time, expiratory time, and respiratory frequency between the eight groups. *Significant difference compared with the baseline condition
for a given variable; †significant difference compared with the continuous positive airway pressure (CPAP) off condition for a given variable: P ⬍ 0.05.
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GENIOGLOSSUS BEHAVIOR WITH INCREASED VENTILATION AND CPAP
Fig. 6. Peak discharge frequency of inspiratory phasic and inspiratory tonic single motor units active in the condition of elevated CO2 compared with baseline
(x-axis). The peak discharge rate of inspiratory phasic units (A) and inspiratory tonic units (B) active in CO2 breaths is plotted against their peak discharge rate
in the baseline condition. Units active in both CO2 and baseline condition are represented by open circles. Units that were recruited and derecruited between
conditions are represented by open squares. Solid circles and squares represent mean ⫾ SE values. The subtle changes in the levels of rate coding can be observed
between the conditions: a slight increase in rate coding can be observed with CO2 compared with baseline in the inspiratory phasic units. Only two of the
inspiratory tonic units did not respond with an increase in rate coding to hypercapnic stimulation (the lower number of units most likely contributed to the lack
of significance). This figure further reveals that the newly recruited units’ [indicated by the squares on the y-axis] mean peak discharge frequency (tonic 22 Hz
and phasic 18 Hz) was lower than the mean discharge frequency of the units that were already increasing their rate coding in response to the CO2 condition (tonic
25 Hz and phasic 22 Hz).
chemoreceptor and mechanoreceptor stimulus manipulation.
Using stimulation of chemoreceptors to increase ventilation
and test motoneuron responses, we have observed that genioglossus inspiratory phasic SMUs significantly increase their
firing rates by ⬃2 Hz. However, the discharge rate does not
increase equally for each class of motor unit, as the inspiratory
tonic motor unit class did not increase. Facilitation of the
motoneuron pool by increased chemical drive to breathe led to
a substantial increase in the number of motor units active.
Accordingly, these data suggest that recruitment of new units is
an important mechanism by which work output of the genioglossus muscle is achieved. Furthermore, with the application
of CPAP, there was a reduction in the firing rates of inspiratory
phasic units and total number of active motor units, despite the
elevated levels of CO2. These data support the influence for a
strong inhibitory role that reduced input from mechanoreceptors has on the output of the hypoglossal motor nucleus
(13, 48).
Contrary to our initial hypothesis, our results indicate that
the different classes of motor units did not modify their
behavior as a single population in response to elevated CO2.
Rather, the peak firing frequencies of the inspiratory phasic and
inspiratory tonic classes of units were altered in a disparate
manner. Interestingly, the five different classes of units did
respond in a homogeneous manner in terms of their percentile
distribution of active units under the eight conditions. Furthermore, for the inspiratory units that were already active, there
was earlier activation of units with CO2, together with delayed
activation of inspiratory modulated units during CPAP.
It is well established that the output of motoneurons depends
on their properties and responses to synaptic inputs (8, 34, 45).
Changing the number of motor units that are active (motor unit
recruitment) and the rates at which motoneurons discharge
action potentials (rate coding) will alter the force that a muscle
exerts (8). However, recruitment and rate coding strategies
between muscles are known to vary throughout the range of the
contraction (8, 10). In the genioglossus muscle during lowlevel contractions, the discharge rates of the motor units are
already high compared with limb and respiratory pump muscles (40, 43). Therefore, it may not seem surprising that the
relative increase in rate coding seems minimal (at ⬃2 Hz) with
moderately high levels of CO2 in the waking state. Therefore,
Table 3. Percent distribution of the different classes of units throughout the protocol
Baseline
CO2
2-cmH2O CPAP
4-cmH2O CPAP
6-cmH2O CPAP
8-cmH2O CPAP
10-cmH2O CPAP
CPAP off
Total
No. of Units
Inspiratory Phasic
Units, %
Inspiratory Tonic
Units, %
Expiratory Phasic
Units, %
Expiratory Tonic
Unit, %
Tonic and Tonic Other
Units, %
63
93
91
82
67
59
42
94
591
59
53
56
59
54
56
45
57
55
21
31
25
23
24
27
31
22
25
3
1
2
4
7
2
0
3
3
10
8
11
11
10
8
10
10
10
8
8
5
4
4
7
14
7
7
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GENIOGLOSSUS BEHAVIOR WITH INCREASED VENTILATION AND CPAP
1947
Fig. 7. Peak discharge frequency of all single motor units active in the condition of elevated CO2 compared with seven conditions (x-axis). Data are from
recordings in the genioglossus muscle from 15 subjects. A–G: the peak discharge rate of units active in CO2 breaths is plotted against their peak discharge rate
in seven conditions. Units active in both CO2 and conditions (baseline, 2 cmH2O, 4 cmH2O, 6 cmH2O, 8 cmH2O, 10 cmH2O, and CPAP off; A–G, respectively)
are represented by open circles. Units that were recruited and derecruited between conditions are represented by open squares. Solid circles and squares represent
mean ⫾ SE values. During baseline, the discharge rate of 63 active motor units is plotted against their peak rate in CO2. Under the hypercapnic condition, an
additional 33 new motor units discharged. In contrast, during the higher CPAP levels (E and F), the number of active units was reduced to below baseline
conditions. At 10 cmH2O, the number of active units was 36% fewer than baseline. The subtle changes in the levels of rate coding can be observed between
the conditions as a slight facilitation with CO2 compared with baseline and progressive inhibition with the increased levels of CPAP pressure. In all panels, the
mean peak discharge frequency of motor units active with CO2, but not the condition compared, was lower than the mean frequency of those units that remained
active.
the genioglossal motor units were consistent with the phenomenon that the newly recruited units (or higher threshold) tended
to discharge at lower peak rates than the earlier recruited
low-threshold units (15). The mechanical impact of a 2.0-Hz
increase in firing is uncertain, particularly because the maximum firing rate (and tetanic threshold) for respiratory stimulation of genioglossus remains unclear.
Baseline vs. CO2. Multiple studies have shown chemoreceptor activation (increased PCO2) to increase the “global” upper
airway muscle activity during hypercapnia in animal models
(e.g., Refs. 5, 9, 46) and human studies (e.g., Refs. 31, 32, 47).
The excitation of hypoglossal motoneurons achieved with
increased ventilation is consistent with the effect produced at
the motoneuron pools of the parasternal intercostals and
scalenes that show more prominent recruitment than frequency
modulation (16). This finding is in contrast to phrenic output in
humans that shows a preferential increase in rate coding with
CO2 stimulation (16). Inspiratory phasic hypoglossal motoneurons discharge for longer proportions of inspiration, whereas
the phasic component of inspiratory tonic motor units does not
exhibit this effect. This difference in behavior between inspiratory classes of motor units suggests that there may be different
drives to these motoneurons, and they may have different
premotor sources (36). Alternatively, intrinsic properties of the
motoneurons themselves, such as size-related properties, or
active neuromodulators, may control or limit increases in
J Appl Physiol • VOL
discharge (8, 34). Our data suggest that the increased excitation
of the hypoglossal nucleus produces increases in rate coding in
the phasic class of motor units, while the tonic motor units do
not receive increases in their already fast discharge rates (see
Fig. 7). There is convincing evidence that mild hypercapnia is
associated with increases in the underlying electrical activity
within the respiratory muscles (16, 36), although how this is
reflected in neuronal discharge patterns is a matter of contention across different motoneuron pools. Recent investigations
concluded that recruitment (or derecruitment) of units is the
predominant manner in which the human genioglossus EMG
signal is increased (or decreased) (30, 51, 52). However, on the
basis of the present study, we argue that a higher stimulus
intensity, in contrast to earlier studies (30, 36), results in a
small increase in rate coding of “phasic” units, together with
the recruitment of previously silent units. This result has
implications for the interpretation of previous work in OSA vs.
control subjects, which showed that OSA patients had an
⬃3-Hz higher rate in the inspiratory phasic motor units, together with an ⬃3-Hz lower rate in the inspiratory tonic motor
units (39). We interpreted this as indicating a lack of evidence
for increased global central drive. However, increases in central drive, based on our present data, may only show trends for
such increases in discharge frequency for inspiratory tonic
units (18), but rather small increases in the frequency of
inspiratory phasic units. This finding suggests that OSA pa-
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GENIOGLOSSUS BEHAVIOR WITH INCREASED VENTILATION AND CPAP
tients may have increased central drive. Based on our new data,
we believe the observed decrease in discharge rates of inspiratory tonic units during the rebreathing condition is due to the
recruitment of more inspiratory tonic units at lower discharge
frequencies (possibly driven by persistent inward currents)
(18). Motor units with persistent inward currents have the
ability for self-sustained, rate-limiting discharge that is induced
after a brief input with mediation through monoaminergic drive
(18). Thus, once inspiratory tonic units are active, additional
synaptic input may minimally affect their discharge rate (18).
Pressure changes on genioglossus muscle respiratory activity.
Both animal and human studies have demonstrated a mechanoreceptor pressure-driven activation of upper airway muscles (13, 19, 20, 29, 48, 50). Given that negative pressure
activates genioglossus through stimulation of mechanoreceptors, we sought to determine the pattern of deactivation of
genioglossus SMUs, which would occur with the application of
positive airway pressure (13). The application of positive
airway pressure in 2-cmH2O increments had a graded response
in inhibiting the activity of units that were active across all unit
classes. This pattern of derecruitment is different from other
situations, such as in sleep onset, where a predominant decrease in only the inspiratory (phasic and tonic) classes of units
has been observed (52). Our findings have implications for
patients using chronic CPAP, since the observed histochemical
changes in the genioglossus (26, 33) with treatment in prior
studies (11) is possibly a result of reduced genioglossal motor
unit activity for extended periods i.e., overnight (e.g., Ref. 17).
Methodological considerations. First, we used eight conditions to record genioglossus activity and assess the responsiveness of the same motor units to increased ventilation and
monitor the graded effects of positive airway pressure over
successive 1-min periods. A 10-Torr increase in PETCO2 was
used as a cutoff for each trial in each subject. However, our
ability to follow the discharge of the same units through the
protocol with increased respiratory drive led us to accept an
increase of 6 Torr in PETCO2 that was associated with a clear
increase in the multiunit signal, where the single-unit activity
could still be monitored. Second, it has been previously observed that genioglossus units can switch between classes in
transitions from wakefulness to sleep onset (52); however, we
note that this “class switching” can also happen in wakefulness, and the strength of the central pattern generators to the
motoneurons may be influenced over time (unpublished observations; see Fig. 3). The main analysis was performed on the
population responses of motor units due to the intermittent
recruitment/derecruitment of the SMUs. The limitation of this
approach was that it did not give us detailed information as to
how the same units were responding across each condition.
Consequently, we performed a subset analysis of the same
genioglossus SMUs across multiple conditions. In this subset
of inspiratory phasic units, we elected not to use the 10-cmH2O
condition, as the small amount of washout of CO2 from the
CPAP may have confounded the analysis. Despite this issue,
we found clear evidence for graded responses with the application of CPAP at 8 cmH2O with a 3.3-Hz decrease in
discharge rates from CO2. The firing times were also markedly
altered with a 22% TI decrease in the duration of discharge at
8 cmH2O compared with hypercapnic stimulation. This observation suggests that, despite a minimal washout occurring at
the highest level of CPAP, meaningful conclusions could still
J Appl Physiol • VOL
be drawn. Finally, we recognize that, although we used standard respiratory stimuli (i.e., CO2 rebreathing and CPAP),
these manipulations are complex (e.g., CO2 could increase
negative airway pressure during hyperventilation and CPAP
can influence end-expiratory lung volume), making definitive
conclusions complex.
Summary and conclusions. In summary, these data provide
new information on how chemoreceptor and mechanoreceptors
affect the activity of genioglossus motor units in humans. The
various classes of genioglossus SMUs do not increase their
firing rates and preinspiratory timing in a uniform manner
across the motoneuron pool. Chemoreceptor activation substantially increased ventilation and the number of motor units
active, suggesting that recruitment remains an important mechanism by which the genioglossus muscle is controlled. The
increase in the discharge rates of inspiratory phasic motor units
is in contrast to the population of inspiratory tonic units, which
show no such increase. Of note, we show that the application
of CPAP reduced the firing rates of inspiratory phasic units and
the total number of active motor units. This finding supports
the influence of mechanoreceptors on hypoglossal motor output, which is capable of outweighing the chemoreceptor excitatory response. These data may have clinical implications for
novel therapeutic targets in OSA.
ACKNOWLEDGMENTS
We thank Karen Stevenson for the recruitment of subjects and all staff at the
Sleep Disorders Program of the Brigham & Women’s Hospital for assistance.
GRANTS
This work was supported by the American Heart Association Grant
0840159N and National Heart, Lung, and Blood Institute Grants R01
HL085188, R01 HL090897, K24 HL093218, and P01 HL095491. J. P.
Saboisky is supported by the American Heart Association Founders Affiliate
Postdoctoral Fellowship Award (0826061D). D. J. Eckert is supported by
Overseas Biomedical (CJ Martin) Fellowship from the National Health and
Medical Research Council of Australia (510392).
DISCLOSURES
D. P. White is the Chief Medical Officer for Philips Respironics. D. J.
Eckert consults for Apnex Medical. A. Malhotra has received consulting
and/or research income from Philips, Apnex Medical, Medtronic, Ethicon,
Pfizer, Merck, SGS, SHC, Itamar, Cephalon, and Sepracor. A. S. Jordan
consults for Apnex Medical. A number of the authors have industry affiliations
related to the treatment of sleep apnea. This study examining physiological
mechanisms was funded via peer review grant agencies.
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