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1 Departments of Medicine and Physiology, University of Toronto, Toronto, Canada M5S 1A8; and 2 Department of Human Anatomy and Physiology, Conway Institute, University College, Dublin 2, Ireland
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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The effects of sleep on the ventilatory responses to hypercapnia have been well described in animals and in humans. In contrast, there is little information for genioglossus (GG) responses to a range of CO2 stimuli across all sleep-wake states. Given the notion that sleep, especially rapid eye movement (REM) sleep, may cause greater suppression of muscles with both respiratory and nonrespiratory functions, this study tests the hypothesis that GG activity will be differentially affected by sleep-wake states with major suppression in REM sleep despite excitation by CO2. Seven rats were chronically implanted with electroencephalogram, neck, GG, and diaphragm electrodes, and responses to 0, 1, 3, 5, 7, and 9% CO2 were recorded. Diaphragm activity and respiratory rate increased with CO2 (P < 0.001) across sleep-wake states with significant increases at 3-5% CO2 compared with 0% CO2 controls (P < 0.05). Phasic GG activity also increased in hypercapnia but required higher CO2 (7-9%) for significant activation (P < 0.05). Further studies in 15 urethane-anesthetized rats with the vagi intact (n = 6) and cut (n = 9) showed that intact vagi delayed GG recruitment with hypercapnia but did not affect diaphragm responses. In the naturally sleeping rats, we also showed that GG activity was significantly reduced in non-REM and REM sleep (P < 0.04) and was almost abolished in REM even with stimulation by 9% CO2 (decrease = 80.4% vs. wakefulness). Such major suppression of GG activity in REM, even with significant respiratory stimulation, may explain why obstructive apneas are more common in REM sleep.
sleep; hypercapnia; control of breathing; vagus nerves
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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DETERMINING THE MECHANISMS underlying the effects of sleep on the control of breathing is relevant to understanding normal respiration and the pathogenesis of sleep-related breathing disorders. In particular, given that CO2 is considered a key component in respiratory regulation, several previous studies in animals and humans have determined the effects of sleep on the ventilatory responses to hypercapnia (e.g., Refs. 7, 40; see Ref. 38 for review). Surprisingly, however, there is little information for responses of the pharyngeal muscles such as genioglossus (GG) to a range of CO2 stimuli across all sleep-wake states. Characterization of such responses is necessary given that the pharyngeal muscles are importantly involved in the maintenance of upper airway patency, and reduced activity and reflex responses in sleep contribute to increased upper airway resistance, hypoventilation, and obstructive sleep apnea (17, 26, 42).
A recent study in humans showed that increasing PCO2 to 6 Torr above eupnea produced significant stimulation of respiratory pump muscle activity in non-rapid eye movement (REM) sleep, as judged by increased tidal volume and pulmonary ventilation, but no GG activation was observed (41). However, the effects of REM sleep on the GG responses to CO2 were not investigated in that study, and responses during wakefulness were studied in only 2 of 18 subjects (41). Badr et al. (2) reported variable degrees of GG activation with a similar level of CO2 stimulation in non-REM (NREM) sleep, but again GG responses in awake and REM sleep were not reported, thereby precluding determination of state effects. A separate study, however, reported that GG could be reliably activated by hypercapnia in awake humans (31). Taken together, evidence suggests that GG responses to CO2 may be decreased in NREM sleep (2, 41), but the magnitude of the decrease from wakefulness and the additional influences of REM sleep have not been determined.
This surprising lack of information on the GG response to CO2 across all sleep-wake states also extends to animal studies. In rats, GG responses to CO2 have been measured in NREM and REM sleep, but responses across a range of CO2 stimuli and direct comparisons with respiratory pump muscle activity were not performed (28). Moreover, no group data, awake or asleep, were presented in that study, with only anecdotal observations reported, and respiratory activities were measured at different ambient temperatures to induce changes in posture. Two studies in cats reported increased GG and diaphragm activities with CO2, but the responses in wakefulness (15) and in NREM sleep (16) were reported separately for different cats, precluding direct comparisons, and the effect of REM sleep was not investigated. Studies in sleep-deprived goats showed that GG responses to progressive hypercapnia were transiently decreased in phasic REM sleep, but overall no significant differences were detected in the GG responses to CO2 across sleep-wake states (36).
Therefore, the present study aims to determine the GG responses to a range of CO2 stimuli across wakefulness, NREM sleep, and REM sleep and to compare these responses to those of the diaphragm. Studies were performed in rats because of their particular suitability for chronic instrumentation and long-term experiments in sleep, as well as their suitability for future studies using stereotaxic techniques to determine central neural mechanisms underlying sleep-specific effects on the control of breathing (e.g., Ref. 22). Given the notion that total motor outflow to respiratory muscle is the sum of respiratory and nonrespiratory inputs, with the latter most affected by sleep mechanisms (19, 33, 47), the present study tests the hypothesis that GG activity would be more suppressed in sleep than that for the diaphragm. The present study also tests the hypothesis that GG activity and the excitatory responses to hypercapnia will be differentially affected by sleep-wake states, with major suppression in REM sleep despite CO2 stimulation. The reason for the latter hypothesis is that we have previously observed periods of significant GG suppression in natural REM sleep despite local excitation of the hypoglossal motor nucleus with applied neurotransmitters (22). This observation suggested that REM sleep mechanisms might be able to override even strong excitatory drives to GG (22). Determining whether respiratory pump and pharyngeal muscle activities are differentially affected by sleep-wake states and CO2-mediated excitation has relevance to the control of breathing, the mechanisms maintaining airway patency, and the pathogenesis of obstructive sleep apnea.
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Study 1: Naturally Sleeping Rats
Studies were performed on seven male Wistar rats (mean weight = 278 g, range = 241-315 g, Charles River). Rats were housed individually, maintained on a 12:12-h light-dark cycle (lights on 0700), and had access to food and water ad libitum. All procedures conformed to the recommendations of the Canadian Council on Animal Care, and the experimental protocol was approved by the University of Toronto Animal Care Committee.Surgical preparation. Sterile surgery was performed under anesthesia induced with intraperitoneal ketamine (85 mg/kg) and xylazine (15 mg/kg). Rats were also intraperitoneally injected with buprenorphine (0.03 mg/kg), atropine sulphate (1 mg/kg), and saline (3 ml, 0.9%). An anesthesia mask (8) was placed over the snout, and the rats spontaneously breathed a 50:50 mixture of room air and oxygen. Any subsequent anesthesia was given by inhalation (halothane, typically 0.2-2%). Effective anesthesia was judged by abolition of the hindlimb withdrawal and corneal blink reflexes. Body temperature was monitored with a rectal probe and maintained between 36 and 38°C (BAS, West Lafayette, IN).
With the rats supine, the ventral surface of GG was exposed via a submental incision and dissection of the geniohyoid and mylohyoid muscles. Two insulated, multi-stranded stainless steel wires (AS631; Cooner Wire, Chatsworth, CA) were sutured bilaterally under direct vision into GG muscle. To record diaphragm activity, two similar wires (AS636; Cooner Wire) were sutured onto the costal diaphragm via an abdominal approach. To ensure adequate electrode placements during surgery, the GG and diaphragm signals were monitored on chart (Grass model 79D polygraph, 7P511 amplifiers) and loudspeaker (AM8 audio amplifier, Grass). Observing tongue protrusion in response to electrical stimulation (0.9-2.0 V) was also used to confirm that electrodes were in GG muscle (11), and this response was also tested after the experiments (see Protocol). GG and diaphragm wires were tunneled subcutaneously to a neck incision, and the submental and abdominal incisions were closed with absorbable sutures. The rats were placed in a stereotaxic apparatus (Kopf model 962, Tujunga, CA) with blunt ear bars. To record the electroencephalogram (EEG), two stainless steel screws (0-80) attached to insulated wire (30 gauge) were implanted in the skull over the frontal-parietal cortex. These electrodes were positioned ~2 mm anterior and 2 mm to the right of bregma and 3 mm posterior and 2 mm to the left of bregma, respectively (20). The reference electrode was placed ~5 mm anterior and 3 mm to the left of bregma. Two insulated, multi-stranded stainless steel wires were sutured onto the dorsal cervical neck muscles to record the neck electromyogram (EMG). At the end of surgery, the electrodes were connected to pins inserted into a miniature plug (STC-89PI-220ABS, Carleton University, Ottawa, Canada). The plug was affixed to the skull with dental acrylic and anchor screws. After surgery, rats were transferred to a clean cage and kept warm under a heating lamp until full recovery, as judged by normal locomotor activity, grooming, drinking, and eating. Rats were given soft food for the first day after surgery, and all rats recovered fully and were not studied before 7.1 ± 1.6 (SD) days (range = 4-8 days). At the time of the experiments, rats weighed 280.6 ± 9.9 g, which was not different from their presurgical weight (P = 0.694, paired t-test).Recording procedures. For recordings, a lightweight shielded cable was connected to the plug on the rat's head. The cable was attached to a counterbalanced swivel, which permitted free movement. All rats were studied in a noise-attenuated, electrically shielded cubicle (EPC-010, BRS/LVE, Laurel, MD) free from interruption. A one-way mirror allowed visual monitoring without disrupting the rat. Rats were placed in an experimental chamber (MD-1500, BAS) and connected to the cable and swivel apparatus the day before the experiments for habituation. The chamber contained food, water, and bedding from the rat's own home cage.
A custom-made lid on the experimental chamber contained a center hole for the cable carrying the electrophysiological signals from the rat. To disperse and mix the air as it flowed into the chamber, the lid also contained three air inlet ports with small computer fans (model FP-108G/DC, 12 V, Commonwealth, driven at 6 V DC). The volume inside the chamber with the bedding and lid in place was ~20.5 liters. Air or CO2 mixtures (see Protocol) were delivered at ~5 l/min after humidification over a water reservoir. CO2 levels were measured continuously (Beckman LB-2) at a flow rate of 500 ml/min with the sampled air being returned to the chamber. The relative humidity and temperature were also measured continuously by a calibrated thermohygrometer probe (model 37950-10, Cole-Parmer Instruments, Vernon Hills, IL) and averaged 42.8% (range = 36.8-53.5%) and 26.1°C (range = 24.2-27.2°C), respectively. Electrical signals were amplified and filtered (Super-Z head-stage amplifiers and BMA-400 amplifiers/filters, CWE, Ardmore, PA). EEG was filtered between 1 and 100 Hz, whereas the neck, GG, and diaphragm EMGs were filtered between 100 and 1,000 Hz. The electrocardiogram was removed from the diaphragm signal by using an oscilloscope and an electronic blanker (model SB-1, CWE). Moving-time averages (time constant = 200 ms) of the neck, GG, and diaphragm EMGs were also obtained (Coulbourn S76-01, Lehigh Valley, PA). Signals were recorded on chart (Grass model 78D polygraph) and computer (Spike 2 software, 1401 interface, CED, Cambridge, UK).Protocol. Experiments were typically performed between 0900 and 1900, i.e., during the time the rats normally sleep. Recordings of sleep-wake states and respiratory muscle activities were made with the rats breathing room air and 1, 3, 5, 7, and 9% CO2 delivered from calibrated pressurized tanks. The different CO2 concentrations were presented in random order to each rat. Rats were returned to room air breathing for at least 20 min between each CO2 concentration.
At the end of the experiment, the rats were reanaesthetized with ketamine (85 mg/kg) and xylazine (15 mg/kg), and confirmation of tongue protrusion in response to electrical stimulation of the GG electrodes was performed (11). After we confirmed tongue protrusion with electrode stimulation, the rats were overdosed with an intraperitoneal injection of pentobarbital sodium (100 mg/100 g).Analyses. Wakefulness, NREM sleep, and REM sleep were determined visually from the chart records in 10 s epochs by using standard EEG and EMG criteria (20). Brief arousals from sleep, lasting from 3 to 10 s, were also identified from the EEG and EMG signals (1). To minimize bias in data analysis, the EEG and neck EMG were scored for sleep-wake states without reference to the GG or diaphragm signals.
GG, neck, and diaphragm EMGs were analyzed in wakefulness, NREM, and REM sleep from periods lasting >30 s. Signals were analyzed for sleep-wake periods that occurred at least 10 min after a switch to each CO2 level. At this time, the rat was judged to be in steady state because equilibration in the chamber to the desired CO2 occurred after ~4 min. EMGs were analyzed each 5 s from the respective moving average signal (above electrical zero) and were quantified in arbitrary units. Electrical zero was the voltage recorded with the amplifier inputs grounded. Each rat served as its own control with all interventions performed in one experiment, thereby allowing for consistent effects of experimental condition (e.g., CO2) to be observed across sleep-wake states within and between rats. GG was quantified as mean tonic activity (i.e., basal activity in expiration), peak activity, and phasic respiratory activity (i.e., peak inspiratory activity and tonic activity). Mean neck muscle activity, diaphragm amplitudes (i.e., phasic respiratory diaphragm activity), and respiratory rates were also calculated for each 5-s period. EEG was sampled by computer at 500 Hz and then analyzed on overlapping segments of 1,024 samples, windowed by using a raised cosine (Hamming) function, and subjected to a fast Fourier transform to yield the power spectrum. The window was advanced in steps of 512 samples, and the mean power spectrum of the EEG signal over each 5-s analysis epoch was calculated. The power contained within six frequency bands was recorded as absolute power and also as a percentage of the total power of the signal. The band limits were
2
(0.5-2 Hz), 1 (2-4 Hz), 
(4-7.5 Hz),
(7.5-13.5 Hz), 
1 (13.5-20 Hz), and
2 (20-30 Hz). Overall, the analysis provided
breath-by-breath measurements of GG and diaphragm activities, and mean
neck and EEG frequencies calculated and averaged in consecutive 5-s
bins. In each rat, these values were then matched to the respective
sleep-wake states to provide a grand mean for each variable in each
state at each CO2 level. Data at arousals from sleep and
transitions between sleep-wake states were excluded from the analysis.
Any respiratory or EEG data with electrical noise or artifact (e.g.,
associated with overt body movements during grooming) were also excluded.
Study 2: Anesthetized Rats With Vagus Nerves Intact and Cut
To determine the role of the vagus nerves in modulating the GG responses to CO2, further studies were performed on 15 urethane-anesthetized rats (1 g/kg ip; mean weight = 287 g, range = 248-350 g). These particular studies were performed under anesthesia because we felt that there would be significant problems with chronic section of the cervical vagus in behaving rats, e.g., with gut motility and regulation of digestion, that would make such experiments untenable. Moreover, techniques for acute reversible blockade of the vagus nerves, e.g., cooling as used in large animals such as dogs (39), are not currently feasible in rats because of their small size. The anesthetized rats were tracheotomized, and the femoral artery and vein were cannulated. Nine of the rats were bilaterally vagotomized, and six were left with the vagi intact. Rats spontaneously breathed a 50:50 mixture of room air and oxygen with additional anesthesia given by inhalation (halothane, typically 0.2-2%) to achieve a high-voltage, low-frequency EEG signal. Body temperature was maintained between 36 and 38°C with a heating pad, and rats received continuous intravenous fluids (0.4 ml/h of a solution of 7.6 ml saline, 2 ml 5% dextrose, and 0.4 ml of 1 M NaHCO3). To ensure consistent head and body position, rats were held in a stereotaxic apparatus in the prone position.Recordings, protocol, and analyses. EEG, GG, and diaphragm EMGs were recorded on chart and computer (see Study 1: Naturally Sleeping Rats above) with blood pressure (DT-XX transducer, Ohmeda, Madison, WI, and PM-1000 Amplifier, CWE) and inspired CO2 concentration (CapStar-100, CWE). Recordings were made with rats breathing room air, 2.5, 5, 7.5, 10, and 12.5% CO2, with each stimulus presented in random order and maintained for at least 6 min to achieve steady state. As a control, at least 6 min of room air breathing before and after each CO2 stimulus was performed. At the end of the experiments, rats were given an overdose of urethane (2 g/kg), and euthanized by intravenous injection of 3-5 ml of saturated KCl solution.
All signals were analyzed in the last minute of room air breathing before the CO2 stimulus, in the last minute of steady-state CO2 stimulation, and in the last minute after removal of CO2. GG and diaphragm EMGs, blood pressure, and EEG were analyzed every 5 s from the respective moving average signal as described above.Statistical Analyses
Analyses performed for each test are included in the text where appropriate. For all comparisons, differences were considered significant if the null hypothesis was rejected at P < 0.05 by using a two-tailed test. When post hoc comparisons were performed after repeated-measures ANOVA, the Bonferroni-corrected P value was used to infer statistical significance. For two-way ANOVA, the factors were sleep-wake states (i.e., wakefulness, NREM sleep, and REM sleep) and CO2 level. The onset of statistically significant GG or diaphragm responses to CO2 was determined within a given sleep-wake state by using one-way ANOVA followed by Dunnett's test for comparisons with a single control (i.e., 0% CO2). The appropriate nonparametric tests were performed for data, which were not normally distributed. Analyses were performed by using Sigmastat (SPSS, Chicago, IL). Results are presented as means ± SE.| |
RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Study 1: Naturally Sleeping Rats
GG recordings.
Electrical stimulation before and after the experiments confirmed that
the electrodes were correctly placed in GG throughout the study (see
METHODS), with further confirmation coming from postmortem
visual examination of the tongue musculature. Tonic GG activation was
prominent during room air breathing in quiet wakefulness, but phasic
respiratory-related GG activity was recruited with added
CO2 (Fig. 1). Despite the
prominent tonic GG activity during room air breathing in wakefulness, a
small degree of phasic respiratory-related GG activity was detected
when GG was observed at a high sensitivity (Fig. 1, and see analyzed
group data in Fig. 2).
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Responses to CO2. Figure 1 shows that, during room air breathing, there were marked reductions in GG activity from wakefulness to sleep such that GG was minimally active except for occasional muscle twitches in REM. In contrast, both the GG and diaphragm are stimulated in hypercapnia, with phasic respiratory-related GG activity persisting in NREM sleep despite the lowered activity compared with wakefulness. Note, however, that REM sleep is associated with a major suppression of GG activity despite CO2 stimulation.
Activation in response to CO2.
Figure 2 shows the mean respiratory responses for the group of seven
rats. Diaphragm activity increased significantly with CO2
in wakefulness, NREM sleep, and REM sleep (P < 0.0001 for all, one-way ANOVA; Fig. 2A). Statistically significant
diaphragm activation, compared with room air controls, was observed in
response to 
5% CO2 in wakefulness and NREM sleep and
3% CO2 in REM (P < 0.05, Dunnnett's
test after one-way ANOVA).
Magnitude of responses to CO2. GG responses to CO2 were significantly different across sleep-wake states (P = 0.029, two-way ANOVA; Fig. 2B). Post hoc tests showed that phasic respiratory GG activity significantly decreased from wakefulness to NREM sleep (P = 0.002) and decreased further from NREM to REM (P = 0.001). In contrast, tonic GG activity did not respond to CO2 (P = 0.654, two-way ANOVA), although there were significant decreases in overall tonic GG activity from wakefulness to sleep (P < 0.002, post hoc paired t-test; Fig. 2D).
Diaphragm responses to CO2 were significantly different across sleep-wake states (P = 0.0001, two-way ANOVA; Fig. 2A). There was a significant decrease in diaphragm activity from wakefulness to NREM sleep (P = 0.011, post hoc paired t-test). However, REM sleep was associated with increased phasic diaphragm activity compared with NREM sleep (P = 0.002; Fig. 2A) and wakefulness (P = 0.049).Respiratory rate.
Respiratory rate varied significantly with CO2 across each
sleep-wake state (P < 0.003 for all, one-way ANOVA;
Fig. 2D) with the onset of a significant response being 5%
CO2 in wakefulness and REM sleep and 1% CO2 in
NREM sleep (P < 0.05, Dunnnett's tests). However, the
effect of CO2 on respiratory rate was different across sleep-wake states (P < 0.0001, two-way ANOVA), and
this was particularly obvious in REM (Fig. 2D); respiratory
rates were higher in REM sleep with room air breathing but were lower
in REM sleep at CO2 levels of 7% (P < 0.05, post hoc paired t-tests).
Neck muscle activity.
Neck muscle activity did not respond significantly to CO2
across sleep-wake states (P = 0.253, two-way ANOVA;
Fig. 3A). However, as
expected, there was a significant independent effect of sleep-wake states on neck muscle tone (P < 0.0001, two-way ANOVA;
Fig. 3A), with reductions in activity from wakefulness to
NREM sleep (P = 0.0004, post hoc paired
t-test) and further reductions in REM sleep
(P < 0.0001).
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Sleep-wake states and EEG power. There was a significant increase in percentage of wakefulness with hypercapnia (P < 0.0001, one-way ANOVA), with effects observed at 7% CO2 compared with room air (P < 0.05, Dunnnett's test; Fig. 3B). Likewise, there was a significant effect of CO2 on REM sleep (P < 0.003, one-way ANOVA), but post hoc testing did not detect significant differences between the different levels of CO2 (P > 0.05, Dunnnett's test). Although there was also a trend to decrease NREM sleep with hypercapnia, this was also not statistically significant (P = 0.113, one-way ANOVA).
There were significant independent effects of both CO2 and sleep-wake states on total EEG power (both P
0.003, two-way
ANOVA; Fig. 3C). As expected, EEG power was greater in NREM
sleep (P < 0.005 compared with waking and REM, post
hoc paired t-tests) but similar between wakefulness and REM
(P = 0.481). There were significant effects of
CO2 stimuli on total EEG power across all sleep-wake states
(each P < 0.03, one-way ANOVAs), with consistent decreases in power, compared with room air, at 5% CO2 for
NREM sleep and 9% CO2 for REM and wakefulness
(P < 0.05, Dunnnett's tests). The reduction in total
power of the NREM sleep EEG in response to CO2 was also
accompanied by a change in the spectral distribution of EEG power with
a shift toward faster EEG frequencies most prominently observed as
increased activity (P = 0.01).
Study 2: Anesthetized Rats With Vagus Nerves Intact and Cut
Significant increases in both diaphragm and GG activities were observed with hypercapnia in the anesthetized rats, with the increases in GG activity being more pronounced with the vagi cut compared with being intact (Fig. 4). Compared with control activity before CO2, significant increases in diaphragm activity occurred at 5% CO2 with both the vagi intact and cut (P < 0.05 for both, post-paired t-tests). In contrast, the onset of statistically significant GG activation occurred at 10% CO2 with the vagi intact but at 2.5% CO2 with the vagi cut compared with room air controls (P < 0.05 for both).
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Blood pressure increased with hypercapnia and reached statistical
significance at 12.5% CO2 with the vagi intact (mean
increase = 9.5 ± 3.0 mmHg, P < 0.05) and at
10% CO2 with the vagi cut (mean increase = 7.4 ± 3.0 mmHg, P < 0.05). As with the conscious rats, there was a significant effect of CO2 on EEG activity
(P < 0.005, one-way ANOVA), with increased observed at high (10%) CO2 with the vagi both cut and
intact (P < 0.05, Dunnnett's tests).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Results of the present study demonstrate that sleep exerts significant modulating effects on GG and diaphragm responses to CO2, with the GG being more suppressed in sleep than the diaphragm and requiring higher percentages of CO2 for significant activation. Another important finding was that GG activity was almost abolished in REM sleep despite strong respiratory stimulation by hypercapnia. In separate experiments in anesthetized animals, we showed that vagal afferent inputs strongly inhibit the GG response to hypercapnia. We suggest that the high percentage of CO2 required for significant GG activation in nonanesthetized animals may, at least in part, be due to the presence of intact vagus nerves. Overall, the results support the concept that sleep exerts profound effects on the activities of muscles, which have dual respiratory and nonrespiratory functions, such as the GG, compared with primary respiratory muscles such as the diaphragm (19, 33). This differential effect of sleep on the GG vs. diaphragm muscle activities and their responses to CO2 has important implications for the maintenance of upper airway patency. Greater suppression of pharyngeal dilator muscle activity in sleep, especially REM sleep, and delayed recruitment with CO2, as observed in this animal model of pharyngeal motor control, would, in humans, predispose to airway narrowing, hypoventilation, and obstructive apneas given that similar mechanisms may be operative (discussed below). In addition, abolition of GG activity in REM sleep and prevention of recruitment despite strong respiratory stimulation may also explain why obstructive apneas are more common in REM sleep.
Major suppression of GG activity in REM sleep. In humans, the transition from wakefulness to NREM sleep is associated with transient reductions in GG activity (44, 53), but clear GG suppression is typically observed in REM sleep (44, 51). Similar suppression of GG activity in REM sleep has been reported in other species, such as rats (22, 28). The present study not only extends these observations but also shows that sleep-wake states significantly modulate GG responses to CO2, with a marked suppression of GG responses in NREM sleep and almost an abolition of responses in REM sleep even with high CO2 levels. The one study preceding ours that did report GG and diaphragm responses to CO2 across all sleep-wake states did so in sleep-deprived goats by using progressive hypercapnia (36). We chose not to use progressive hypercapnia because of inherent problems associated with changes in sleep-wake states during the re-breathe. This problem increased within and between test variability and led to studies in the same goat being performed on different days (36). This increased variability of responses, and the absence of a repeated-measures design, may have contributed to an inability to detect overall differences in the GG responses to CO2 across sleep-wake states in that study (36). Sleep deprivation itself may have also altered GG and diaphragm responses to hypercapnia (27, 50), further complicating data interpretation.
We did not measure arterial blood gases in this study because we wanted to avoid chronic arterial catheterization and repeated blood withdrawals. Accordingly, our only measure of hypercapnic stimulation was inspired CO2 levels. It is possible, therefore, that at any given level of inspired CO2 the actual arterial PCO2 may have been higher in REM sleep, compared with NREM, due to lesser ventilation (Ref. 38 and discussed below). If so, however, it is even more notable that the abolition of GG activity occurs in REM sleep despite this potential additional hypercapnia, an observation that reinforces the concept of major suppression of GG activity in REM sleep despite ventilatory stimulation. There exists considerable debate as to the mechanisms underlying the sleep-related suppression of GG activity, especially as to the relative roles of postsynaptic inhibition and disfacilitation of hypoglossal motor output in REM sleep (see Refs. 18 and 24 for reviews). Much of this debate arises from the applicability of the mechanisms determined in a pharmacological model of REM sleep evoked by cholinergic stimulation of the pontine reticular formation in reduced animal preparations (12, 25, 52). Although the brain stem mechanisms underlying GG suppression were not determined in this study, future studies using in vivo microdialysis of the hypoglossal motor nucleus in naturally sleeping rats (22) can be used to determine the neural processes underlying the major influence of sleep-wake states on the GG responses to CO2 and the relative roles of inhibition and disfacilitation using techniques not feasible in humans. Although hypoxia is recognized to decrease metabolic rate in rodents (29), the effects of hypercapnia on metabolism are less clear, with small increases, decreases, or no change reported (see Refs. 21, 29, 43, and references therein). Given these observations, it is unlikely that our results were significantly affected by potential changes in metabolism, but this cannot be definitively determined in this study because neither body temperature nor metabolism was measured.GG and diaphragm recruitment with CO2. Another major finding from this study was that higher CO2 levels were required for significant GG activation compared with the diaphragm. This difference in response pattern led to an apparently linear CO2 response for the diaphragm across sleep-wake states but a nonlinear response for GG (Fig. 2B). Similar differences in response pattern between the diaphragm and GG with CO2 have been observed in cats (15, 16), goats (36), and infants (4, 6). Of relevance, however, these results in animals and infants differ from those observed in adult humans whose GG activity increases linearly with the diaphragm in response to CO2 (31).
A possible mechanism to explain this difference in GG responses to CO2 between animals and infants compared with adult humans may be the influence of the vagus nerves on hypoglossal motor output. Previous studies (Ref. 14 and references therein) have suggested that the vagus nerves exert greater influence on respiratory motor output in animals and infants. Because vagal afferent activity inhibits hypoglossal motoneurons (45), GG responses to CO2 may therefore be restrained by the increased vagal feedback associated with increased ventilation in hypercapnia. To address this possibility, we performed separate studies in anesthetized rats in which the vagus nerves were either intact or cut. These particular studies were performed in anesthetized rats because of concerns with chronic section of the cervical vagus in behaving rats and because available techniques for acute reversible vagal blockade are currently not feasible in rodents (see METHODS). In these anesthetized rats, the diaphragm responded linearly with CO2 as it did in the conscious rats, but cutting the vagus nerves markedly reduced the CO2 level at which the onset of statistically significant GG activation occurred (Fig. 4B vs. 4D). The relatively high level of CO2 required to stimulate GG in the anesthetized rats with the vagi intact was somewhat surprising, although it was, nevertheless, similar to the level required in the sleeping rats (Figs. 4B vs. 2B). Also, our results in anesthetized rats are in full agreement with Fukuda and Honda (9, 10), who showed that the vagus nerves markedly suppress motor output to GG, but not to the diaphragm, in response to CO2, such that GG responses are variable and inconsistent in hypercapnia with the vagi intact. In an attempt to produce similar degrees of anesthesia across the experiments, it is possible that the presence of a stable high-voltage, low-frequency EEG (i.e., a relatively deep level of anesthesia) may have hindered our ability to stimulate GG until relatively high levels of hypercapnia. Surgical levels of anesthesia (i.e., no response to foot pinch or painful stimulus) can be produced with a mixed frequency or even a desynchronized EEG pattern, and so a lesser state of anesthesia may have increased GG activity and excitability in response to CO2 (46). For example, in anesthetized rats, Fuller et al. (11) observed GG responses at lower levels of CO2 (7%) than those we observed in the present study. It is possible that differences in other physiological variables between the vagus-intact and -cut conditions in the anesthetized rats could account for the apparent change in CO2 levels required for the onset of significant GG activation. However, the increase in blood pressure with CO2 was similar with the vagi intact and cut, making this mechanism unlikely to explain the difference in GG responses. In addition, the shift to increased activity from a background of high-voltage, low-frequency EEG was also similar in the anesthetized rats with the vagi either intact or cut,
suggesting that arousal mechanisms are also not likely to be involved
in the different GG responses. Nevertheless, the observation of a
change in activity with hypercapnia is of interest given that the
EEG is rarely measured during CO2 responses in anesthetized animals despite accumulating evidence that multiple sites involved in
brain arousal also act as CO2 sensors (3, 13, 30, 48, 49). It remains to be determined what role such classically nonrespiratory areas involved in brain arousal and CO2
sensation play in determining ventilatory responses to hypercapnia and
whether this contribution is greater for muscles such as GG compared
with the diaphragm.
The observed difference in CO2 recruitment for the GG and
diaphragm is potentially important because earlier recruitment of the
diaphragm in hypercapnia may promote upper airway instability and
predispose it to airway obstruction. This effect may be especially problematic in infants, compared with adults, if the inhibitory effects
of the vagus nerves are responsible for limiting GG activation in hypercapnia.
Diaphragm activity across sleep-wake states. Results of the present study show that diaphragm activity was increased in REM sleep during both room air breathing and hypercapnia (Fig. 2A). This result, coupled with the higher respiratory rates in REM sleep (Fig. 2D), suggests that REM is a state of heightened respiratory drive. These observations are consistent with those studies showing periods of increased activity of medullary inspiratory neurons in REM sleep (32) and increased diaphragm activity in cats (23, 34, 35), dogs (19), and humans (5).
This concept of a heightened drive to primary respiratory muscles in REM sleep (33) does not conflict with the marked diminution of ventilation and hypercapnic ventilatory responses typically observed in REM sleep in animals and humans (e.g., Refs. 7, 38, 40). Indeed, since total pulmonary ventilation comprises the coordinated activation of the diaphragm and chest wall muscles, especially when stimulated by CO2, suppression of intercostal and postural muscle tone in REM sleep (37) would lead to increased rib cage compliance and contribute to decreased transpulmonary pressures on inspiration (34). These effects, coupled with increased upper airway resistance (17) caused by pharyngeal muscle hypotonia, as observed in this and other studies (e.g., Refs. 44, 51), likely produce the overall decrease in ventilation for a given CO2 in REM sleep. In summary, the present results support the concept that REM sleep leads to marked excitation of central respiratory drive and diaphragm activity and to marked suppression of motor output to GG, which occurs even in the presence of strong respiratory stimulation produced in hypercapnia. Although central neural mechanisms cannot be determined from this study, the results also support the suggestion that sleep mechanisms exert profound effects on muscles such as GG, which receive dual respiratory and nonrespiratory inputs, whereas primary respiratory muscles, such as the diaphragm, are less affected by sleep mechanisms. Such preferential effects of sleep, especially REM sleep, on pharyngeal muscle activity, even in the presence of significant respiratory stimulation as observed in this animal model, would predispose humans to airway instability and obstructive sleep apneas.| |
ACKNOWLEDGEMENTS |
|---|
This work was supported by funds from the Canadian Institutes of Health Research (CIHR) Grant MT-15563. R. L. Horner is a recipient of a CIHR Scholarship.
| |
FOOTNOTES |
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Address for reprint requests and other correspondence: R. L. Horner, Rm. 6368 Medical Sciences Bldg., 1 Kings College Circle, Toronto, Ontario, Canada M5S 1A8 (E-mail: richard.horner{at}utoronto.ca).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
10.1152/japplphysiol.00855.2001
Received 14 August 2001; accepted in final form 25 October 2001.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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), NREM (
), and REM sleep
(
). A: responses of phasic Dia to
CO2. B: respiratory rate responses to
CO2. C: phasic GG responses to
CO2. D: tonic GG responses to
CO2. EMG values are displayed in arb units from their
respective MTA signals. Note the small but measurable degree of phasic
respiratory-related GG activity in wakefulness but not sleep unless
driven by CO2. Data are means + SE. See
RESULTS for further details.
