The human genioglossus response to negative airway pressure: stimulus timing and route of delivery

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1 288 Exp Physiol 93.2 pp Experimental Physiology The human genioglossus response to negative airway pressure: stimulus timing and route of delivery Liam S. Doherty 1, John P. Cullen 1, Philip Nolan 1,2 and Walter T. McNicholas 1,2 1 Respiratory Sleep Disorders Unit, St Vincent s University Hospital, Dublin, Ireland 2 Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Ireland The genioglossus reflex response to sudden onset pulses of negative airway pressure (NAP) in humans is reported to occur more commonly at end rather than onset of expiration when delivered via a mouthpiece. We examined whether this response was modulated by the route of stimulus delivery throughout the respiratory cycle. The genioglossus surface EMG (GGsEMG) response to NAP delivered randomly throughout the respiratory cycle was measured in a set of experiments: (i) 4 stimuli of NAP at 5, 7.5 and 1 cmh 2 O applied to eight healthy, awake, supine males via nose-mask; and (ii) 6 stimuli of 7.5 cmh 2 O NAP applied to 15 subjects via both nose-mask and mouthpiece in random order. Despite similar pressure changes being detected in the epiglottis during both routes of stimulus delivery, far lower pressure changes were measured at the nasal choanae during mouthpiece compared with nose-mask delivery. There were no significant differences between the responses during any phase of respiration, nor when NAP was delivered via nose-mask or mouthpiece. We conclude that the sensitivity of the GGsEMG response to NAP in humans does not vary significantly with phase of respiration or route of breathing. (Received 25 July 27; accepted after revision 12 October; first published online 19 October 27) Corresponding author W. T. McNicholas: Department of Respiratory Medicine, St Vincent s University Hospital, Elm Park, Dublin 4, Ireland. walter.mcnicholas@ucd.ie A reflex response of upper airway muscles to sudden onset negative airway pressure (NAP) has been demonstrated in humans (Horner et al. 1991). It has been proposed that this reflex maintains and/or re-establishes upper airway patency in response to upper airway collapsing pressures. In anaesthetized rabbits (Woodall et al. 1989), this reflex response is greater when applied during inspiration than during expiration and, on further analysis, greater during the early phase of inspiration than in late inspiration. In awake humans, genioglossus (GG) reflex activity has been successfully elicited both in end-expiration and in early inspiration (Horner et al. 1991; Wheatley et al. 1993). However, one report examining only expiration in humans suggested that this reflex response to NAP was modulated according to time of stimulus delivery, i.e. strongest at end-expiration (Tantucci et al. 1998). Theoretically, this response would be in keeping with a reflex-mediated response that is particularly robust during the most vulnerable phase of respiration, i.e. just before the upper airway is exposed to the collapsing negative pressures of inspiration. Preliminary experiments in this laboratory (L.S. Doherty, J.P. Cullen, P. Nolan & W.T. McNicholas, unpublished data) failed to find a difference in GG reflex activity during any phase of respiration when stimuli were delivered randomly throughout respiration at a variety of negative pressures. One possible explanation for this finding is that stimuli were delivered via nosemask, whereas in Tantucci s study stimuli were delivered via mouthpiece. Theoretically, stimuli delivered via nosemask could dissipate in the nasal pharynx and therefore transmit poorly to the epiglottis. In contrast, by the oral route there would be no such impediment to epiglottic delivery of negative pressure. This argument is all the more reasonable when it has been previously shown that negative epiglottic pressure significantly correlates with genioglossus activity across inspiration (Pillar et al. 21). Thus, the first objective of this study was to record the effect of NAP stimuli on genioglossus activity throughout inspiration as well as expiration and, second, to determine whether route of delivery influenced any possible respiratory modulation of this reflex. DOI: /expphysiol

2 Exp Physiol 93.2 pp Genioglossus response to negative airway pressure 289 Methods Ethical approval Fifteen subjects, aged 24.8 (3.2) years [mean (s.d.)] and with body mass index 25.2 (4.5), were recruited from the medical staff of the hospital. Written informed consent was obtained, and the Ethics committee of St Vincent s University Hospital approved the protocol, which conformed with the Declaration of Helsinki. Only male subjects were selected in order to avoid any gender influence on genioglossus activity (Popovic & White, 1995; Pillar et al. 2). All were healthy non-smokers with no history of otolaryngological, respiratory or sleep disorders. Subjects were instructed to avoid alcohol and caffeine on the day of the study. Mouthpiece Each subject had their genioglossus electromyogram (GGsEMG) recorded using bipolar surface electrodes embedded in a dental impression material, modified from a design by Doble et al. (1985; Fig. 1). Dental impression material was fitted around an athletic mouthguard and, while the material was still soft, subjects placed the dental material over their bottom teeth until it was closely apposed to the surface of the genioglossus muscle. Electrodes were then sewn into the impression material in a unilateral configuration 2 mm posterior to the tip of the mouth-guard, perpendicular to the muscle fibres and 5 mm apart. The last 2 mm of wire was bared and coated with silver chloride to increase signal amplitude. An earth electrode was placed on the subject s forehead. To confirm that no change in electrode position occurred during stimulus delivery, the GGsEMG signal was checked before and after each experiment by asking each subject to perform a maximal voluntary contraction of the GG. This was achieved by asking subjects to push their tongue as hard as possible against the mouthpiece. Negative pressure circuit A negative pressure circuit was designed as follows (Fig. 2). Subjects breathed spontaneously either via nose-mask (Respironics Inc., Murrysville, PA, USA) with their mouth shut or via mouthpiece with a nose-clip (P.K.Morgan PLC, Rainham, Kent, UK). An inspiratory and an expiratory hose were both connected to the atmosphere by one-way rebreathing valves (Hans Rudolf, Inc., Kansas City, MO, USA). Attached to the inspiratory hose was a solenoid valve which, when open, could connect the subject to an air-chamber, maintained at 5 cmh 2 O air pressure by a motor, such that the rise time ( 9% of desired pressure target) was reached within 2 ms. On activation of the solenoid valve, the subject was exposed to a 3 ms duration pulse of negative air pressure. Excess negative airway pressure was dispensed with by placing a springloaded reversed PEEP valve (Vital signs Inc., Totowa, NJ, USA) of desired pressure close to the mask. To avoid the noise of stimulus delivery eliciting a startle reflex, the tubing connecting the nose-mask or mouthpiece to the rest of the circuit was fed out of a window of the subject s bedroom and thenfitted to the negative pressure generator and solenoid valve. Equipment and measurements Respiration was monitored by the signal produced by Respitrace bands (Respiband Transducer, Ambulatory monitoring Inc., Ardsley, NY, USA). The Respibands were not calibrated for volume. Airflow was monitored by a 4 l reservoir vacuum Solenoid valve inspiration One-way valves expiration Figure 1. A genioglossus surface electrode fitted with one bipolar pair of electrodes coated with silver chloride and embedded into the dental material Electrodes were sewn 2 mm posterior to the tip of the mouth-guard, perpendicular to the muscle fibres and 5 mm apart. This carrier was worn over the lower teeth, ensuring a close approximation between the electrodes and the underlying genioglossus muscle fibres. Spring-loaded reversed PEEP valve Nose mask or face mask Figure 2. Schematic diagram of negative pressure circuit Subjects inspired and expired normally via a non-rebreathing valve. A solenoid valve, when switched, exposed the subject to negative pressure maintained by a vacuum-filled reservoir. A reversed positive end-expiratory pressure (PEEP) valve placed close to the mask limited negative pressure to a desired level.

3 29 L. S. Doherty and others Exp Physiol 93.2 pp pneumotachograph (Fleisch, Inc., Lausanne, Switzerland) attached to the mask. Mask pressure was recorded via a flexible polyethylene tube connected to a differential pressure transducer, calibrated beforehand with a handheld micromanometer (Digitron P2 S). The EMG signal was amplified (CWE, Inc., Ardmor, PA, USA), filtered (bandpass 3 and 1 Hz) and recorded on computer for later analysis. Data were recorded using a computer-based data capture/signal averaging system (in the first experiment MP1 Hardware and Acqknowledge Software, BIOPAC systems, Santa Barbara, CA, USA was used and in the second experiment Spike2; Cambridge Electronic design Ltd, Cambridge, UK was used). In a third experiment in six subjects, upper airway pressure was determined by the passage of two pressure catheters calibrated simultaneously beforehand. One nostril was first anaesthetized using topical 2% lignocaine. The epiglottic pressure catheter was passed via the anaesthetized nostril until it could be visually inspected passing below the uvula towards the epiglottis (approximately 17 cm). The nasal choanal catheter was passed via the same nostril medial to the first catheter (approximately 9 cm). Both catheters were fixed in place by taping them to the nose. A bias flow through the catheters was not used despite the possibility of blockage of the catheters with secretions. Study protocol (i) Experiment 1. Eight subjects remained awake, supine on a bed and breathed spontaneously via a nose-mask (Respironics Inc.) with their mouth shut. Forty stimuli at each pressure level of 5, 7.5 and 1 cmh 2 Owere delivered in pseudo-random order 3 s apart; to explain this, if the average respiratory rate is breaths min 1 with variations owing to swallows, sighs and coughs, it then follows that stimuli every 3 s would land randomly during any phase of the respiratory cycle, i.e. a pseudorandom delivery. (ii) Experiment 2. Fifteen subjects remained awake, supine on a bed and breathed spontaneously either via a nose-mask (Respironics Inc.) with their mouth shut or via a mouthpiece with a nose-clip (P.K.Morgan). Sixty stimuli at 7.5 cmh 2 O were delivered via the nose-mask or mouthpiece, again in pseudo-random order 3 s apart. Therefore, a total of 12 stimuli were delivered with 6 stimuli delivered via each route of breathing. In six of the subjects, the experiment was repeated with route of breathing in reverse to avoid an order effect. (iii) Experiment 3. A further six subjects repeated the protocol in experiment 2 with 3 stimuli at 1 cmh 2 O via both routes of stimulus delivery, i.e. 6 stimuli. During this experiment, pressure was also recorded simultaneously at the level of the nasal choanae and epiglottis. Analysis Timing of the stimulus was calculated retrospectively by analysis of the pneumotachograph signal and expressed as the time the stimulus was delivered in that phase of respiration as a percentage of the average time spent in that phase. All breaths that coincided with a cough, swallow, sigh or stimulus were discarded from analysis. Stimuli were grouped into one of four different time bins in inspiration and expiration, i.e. < 25, 25 5, 5 75 and > 75% inspiration or expiration. The GGsEMG signal was amplified,filtered, bandpassed and rectified. For each subject, the mean GGsEMG activity in each time bin, 8 12 ms pre- and post-onset of stimulus, was calculated. Thegroup medianggsemg activityin each specific time bin was then used for analysis. Reflex activity was defined either as a ratio (GGsEMG activity 8 12 ms post-onset of stimulus divided by the rectified activity 8 12 ms pre-onset of stimulus) or as a change (GGsEMG activity 8 12 ms post-onset of stimulus minus the rectified activity 8 12 ms pre-onset of stimulus). Statistics Data were expressed either as mean (s.e.m.) if normally distributed, or otherwise as median (quartiles). Data were log-transformed prior to statistical analysis to correct skewness or homogenize variance. One-way ANOVA followed by a non-parametric implementation of the Student Newman Keuls post hoc test was used for statistical comparisons of GGsEMG responses across respiration. A two-way ANOVA was employed for comparisons between routes of stimulus delivery. A P value less than.5 was taken as significant. Results No subject reported any major discomfort throughout the experiment and none admitted or was observed falling asleep. The negative pressure measured at the mask differed slightly from the target pressures. In the first experiment, a target pressure of 5 cmh 2 O was measured as a mean of 5.66 cmh 2 O(s.d. 1.37), target 7.5 cmh 2 O was measured as 7.75 cmh 2 O(s.d. 2.2), and target 1 cmh 2 O was measured as cmh 2 O (s.d..7). The rise time ( 9%) was less than 2 ms but was associated with a small overshoot. Figure 3 shows representative raw data, demonstrating a typical genioglossus reflex response to both nose-mask and mouthpiece delivery of 1 cmh 2 O with the subsequent pressure changes measured at the nasal choana and the epiglottis. Figure 4 illustrates phasic genioglossus activity during both nose-mask and mouthpiece breathing.

4 Exp Physiol 93.2 pp Genioglossus response to negative airway pressure 291 Experiment 1 Negative airway pressure of 5 cmh 2 O increased GGsEMG by 42 (18 139)% (median, range; P <.5) in inspiration and 45 (3 145)% in expiration, while 1 cmh 2 O also significantly increased (P <.1) GGsEMG by 63 (3 146)% in inspiration and 45 ( 135)% in expiration. The GGsEMG response to NAP did not change with increasing pressure (P <.17 for response expressed as a ratio and P =.8 for response expressed as difference by ANOVA and Student Newman A Raw GGsEMG B P choa P epi P mask P choa P epi P mask Raw GGsEMG sec 5 seconds Figure 3. Example of the genioglossus surface EMG response to sudden onset negative pressure at 1 cmh 2 O delivered via nose-mask (A) and mouthpiece (B) Abbreviations: GGsEMG, genioglossus surface electromyogram; P choa, choanal pressure; P epi, epiglottic pressure; and P mask, mask pressure. Keuls post hoc testing on log-transformed data), in the response obtained in inspiration compared with expiration (P <.93 for response expressed as a ratio, P =.49 for response expressed as difference), nor when the responses in the different subdivisions of inspiration and expiration were compared (P <.77 for response expressed as a ratio, P =.15 for response expressed as difference). Experiment 2 There was no significant difference in either tonic or phasic GGsEMG activity, or in the length of time spent in inspiration and expiration when breathing via mouthpiece or nose-mask. Although this experiment was not powered to detect significant differences between routes of stimulus delivery, there was a non-significant trend for a smaller GGsEMG response to NAP when delivered via the mouth compared with responses to stimuli applied via the nose (P =.32). This is more obvious when the response is expressed as difference in activity than the ratio of poststimulus to prestimulus activity (Fig. 5). There was no statistically significant effect of stimulus timing within the respiratory cycle on the magnitude of the GGsEMG response to NAP for either oral or nasal stimuli in this series (P >.16, two-way ANOVA followed by Student Neuman Keuls post hoc testing). Experiment 3 Table 1 illustrates the resulting transmitted pressure changes at the nasal choanae and the epiglottis when 1 cmh 2 O negative pressure is applied via both routes of delivery (P <.1). During nose-mask delivery, regardless of phase of respiration, a lower change in pressure was measured at the nasal choanae and lower still measured at the epiglottis after 1 ms had elapsed (Table 1). In contrast, during mouthpiece delivery, the pressure change detected in the epiglottis was similar to that for NAP stimuli delivered through the nasal airway (P <.17, two-way ANOVA), but minimal transmission of the pressure change to the nasal choanae. Although negative airway pressure delivered to the epiglottis did Genioglossus EMG, mean, SD nose-mask Mean Phasic EMG mouth-piece Figure 4. Graph of phasic genioglossus activity during nose-mask and mouthpiece breathing inspiration expiration

5 292 L. S. Doherty and others Exp Physiol 93.2 pp Table 1. Group pressure changes (in cmh 2 O) measured 1 ms post-stimulus onset at the level of the mask (Pmask), nasal choanae (Pchoa) and epiglottis (Pepi) Nose-mask Mouthpiece Inspiration Expiration Inspiration Expiration P mask 1.6 (.5) 11.2 (.6) 1.5 (.5) 9 (1.8) P choa 5.8 (.8) 7.1 (1.1) 1.6 (.6) 1.3 (.8) P epi 2.7 (.4) 2.1 (.8) 3.8 (1.) 2.8 (1.3) The stimulus applied was 1 cmh 2 O sudden onset negative pressure, delivered during different phases of respiration and via different routes (mouth and nose). Values are presented as means (S.E.M.). not deviate significantly between stimuli at any phase of respiration, the most negative pressures occurred at onset of inspiration and least at early expiration (Fig. 6). Discussion The novel findings in this paper are: (i) the lack of modulation of the GGsEMG response in men not only throughout expiration but also throughout inspiration; and (ii) route of delivery of stimulus does not change this lack of modulation but certainly alters pressure transmission to the nasopharynx. Methodological issues may account for this contrary view. Since the GG exhibits both phasic and tonic activity, the separation of increased reflex motor activity from cyclical activation for analysis purposes becomes very difficult (Remmers et al. 1978; Strohl et al. 198). We feel that quantifying this reflex response as a percentage ratio of maximal muscle activity at rest would make the results vulnerable to the influence of phasic activity, i.e. the biggest reflex response would occur at end-expiration/ early inspiration. We chose to compare EMG activity 8 12 ms post-stimulus to that period 8 12 ms prestimulus, expressed both as a ratio and as a difference in activity. We felt that a time gap of 16 ms between the two periods of EMG activity for comparison was much less likely to be influenced by this phasic EMG activity, although we concede this may not avoid phasic influence completely. A 7 ratio ggemg response 8-12msec post/pre stimulus i1 i2 i3 i4 e1 e2 e3 e4 NOSE-MASK MOUTH-PIECE Respiratory phase B Figure 5. The median (quartiles) genioglossus surface EMG response to 7.5 cmh 2 O negative airway pressure delivered via the nose and mouth during the respiratory cycle A illustrates the response expressed as a ratio of activity post-stimulus to that prestimulus. B illustrates the response expressed as the difference in activity 8 12 ms post-stimulus minus activity 8 12 ms prestimulus. Change in ggsemg response 8-12ms post-stimulus minus 8-12ms prestimulus I1 I2 I3 I4 E1 E2 E3 E4 Respiratory Phase NOSE-MASK MOUTH-PIECE

6 Exp Physiol 93.2 pp Genioglossus response to negative airway pressure 293 By not matching the transmitted pressure to the epiglottis, post-stimulus delivery lessens our ability to comment on the possible respiratory modulation (or lack of) of this reflex. However, this argument also applies to the validity of the conclusions of a paper by Tantucci and coworkers, i.e. this reflex response is most robust at end-expiration (Tantucci et al. 1998). We attempted to explore this further in our third experiment, by measuring airway pressure at the level of the nasal choanae and epiglottis during stimulus delivery. We found that although negative airway pressure delivered to the epiglottis did not deviate significantly between stimuli at any phase of respiration, the most negative pressures occurred at onset of inspiration and least at early expiration (Fig. 6). It then follows that if delivered pressure can be modulated by respiration, then so can the genioglossus reflex response to this pressure. While Tantucci et al. (1998) did not make simultaneous measurements of airway pressure, it is also possible that improved transmission of the pressure stimulus along the length of the airway in the seated position allowed them to detect a respiratory modulation of the GG response to NAP which we could not detect in subjects studied in the supine position. Contrary to this argument, GG peak inspiratory and tonic expiratory EMG has been shown to increase from the sitting to lying position, making the reflex response seem more likely (and relevant) while supine (Douglas et al. 1993; Mathur et al. 1995; Mortimore et al. 1995). Furthermore, lower lung volumes occur in the supine than in the seated position. Stanchina and coworkers demonstrated that reduced lung volumes led to increased genioglossus muscle activation (Stanchina et al. 23). This may possibly account for an unchanged reflex response throughout respiration whilst supine in our study and no detectable reflex activity at early expiration in Tantucci s study while in the seated position. We did not find any influence of route of delivery on the respiratory modulation of this reflex. It was noted that there was a non-significant increase in reflex response when delivery was via nose-mask rather than mouthpiece. This was despite similar levels of pressure reaching the epiglottis via both routes of stimulus delivery. This may possibly reflect the abundance of negative pressure sensory receptors located in the nasopharynx, which are poorly accessed during mouthpiece delivery (White et al. 1998). However, the difference in magnitude of response was not an intended objective, and the experiment was not appropriately powered to reach firm conclusions. Retrospectively, we calculated that 8 subjects would have been required to have an 8% power to detect a two-sided 5% significance level. When pressure was applied via nose-mask, the change in epiglottic pressure was less than the change in nasal choanal pressure, this difference being exaggerated in expiration (Fig. 6B). This finding suggests that either the nose-mask NAP stimuli caused significant narrowing of the airway, more so during expiration, or that the upper A Pressure (cmh 2 O) i1 i2 i3 i4 e1 e2 e3 e4 Phase of respiration P choa P epi P mask B Pressure (cmh 2 O) i1 i2 i3 i4 e1 e2 e3 e4 Phase of respiration P epi P choa P mask Figure 6. Group mean (S.E.M) pressure measured at mask (P mask ), nasal choanae (P choa ) and epiglottis (P epi ) during respiration at 1 ms post-stimulus when 1 cmh 2 O sudden onset negative pressure stimuli were delivered at different times in the respiratory cycle via mouthpiece (A) and nose-mask (B) In this graph, the respiratory cycle has been divided into four equal phases of inspiration i and four equal phases of expiration e.

7 294 L. S. Doherty and others Exp Physiol 93.2 pp airway was already narrowed because of expiration. When NAP was applied via mouthpiece, there was very limited transmission of pressure to the nasal choanae, presumably indicating velopharyngeal closure (Fig. 6A). The change in pressure in both the nasal choanae and the epiglottis was again less in expiration. Application of sudden onset negative pressure was associated with a transient pressure overshoot. This was an inevitable consequence of achieving a near-square pressure wave with a 9% rise time of less than 2 ms, similar to that seen in the original study by Horner et al. (1991). The very rapid pressure change was to ensure an unequivocal reflex latency period and to facilitate calculation of the EMG response by avoiding the influence of volitional GG activation. This very transient pressure overshoot did not alter during any phase of respiration, and no overshoot was measured at the epiglottis in those six subjects that had this pressure recorded (Fig. 3). No difference in GGsEMG reflex activity was seen during incremental changes in NAP delivery. Unfortunately, epiglottic pressure was only recorded at one delivered NAP, namely 1 cmh 2 O. Quite possibly, the transmitted pressure to the epiglottis did not vary during the other delivered NAP values of 5 and 7.5 cmh 2 O, accounting for the lack of differences. We recorded from the GG muscle using surface electromyography, and found on-going tonic activity with minimal phasic inspiratory modulation, as has been reported by other investigators using surface electrodes (Doble et al. 1985; Horner et al. 1991). It is known that respiratory modulation of GG activity is different for different motor units and that there are regional variations in the respiratory modulation in GG activity (Tsuiki et al. 2; Eastwood et al. 23; Saboisky et al. 26). It is possible that the location of our surface electrodes selected a region of the genioglossus muscle where there was minimal respiratory modulation of muscle activity, and therefore minimal respiratory modulation of the reflex response to NAP. If the GG response to NAP was recorded using intramuscular electrodes from areas with marked phasic inspiratory activity, the reflex response might exhibit variations within the respiratory cycle. The recording of minimal phasic activity at baseline is a limiting factor in this study, and the possibility that different regions of the genioglossus muscle are differentially controlled warrants further investigation. Stimuli were delivered pseudo-randomly throughout respiration at 3 s intervals. While subjects could possibly have anticipated the stimulus by counting, none reported this, all were told that stimuli were randomly delivered, and responses were still detected in each subject by the end of the experiment. Although the presence of the genioglossus reflex response to sudden onset negative pressure in man is not disputed, the physiological role of this reflex has yet to be fully understood. Previous studies employing this reflex often used supraphysiological negative pressures ( 1 to 25 cmh 2 O), whereas in awake normal humans, pressure swings are generally less than 4 cmh 2 O. The pressure wave applied was also non-physiological. A square-wave pulse applied from the nose or mouth bears little relationship to the graded change in intraluminal negative pressure, generated from within the thorax, which occurs in response to a narrowed or obstructed airway. The lack of respiratory modulation of this reflex in men may not be surprising for several reasons. In a clinical context, tonic and phasic GG activity was reduced when tracheostomized obstructive sleep apnoea patients switched from nasal to stomal breathing (Malhotra et al. 2). This is in keeping with a reduced negative pressure stimulus, at physiological pressures, delivered to the upper airway. Exposure of the upper airway to sustained negative pressure in animals causes an increase in tonic pharyngeal dilator muscle activity in expiration as well as inspiration (Mathew et al. 1982; van Lunteren et al. 1984). In humans, upper airway obstruction has been observed to occur in expiration as well as inspiration (Lorino et al. 1998; Schneider et al. 2; Lofaso et al. 21). In tracheostomized patients with obstructive sleep apnoea, Schneider et al. 22) demonstrated only a modest difference in upper airway collapsibility in inspiration compared with end- and peak expiratory time. Furthermore, a number of studies have recorded a tight correlation between GGsEMG activity and pharyngeal pressure changes, independent of any changes in airflow effects, chemostimulation, upper airway resistance and respiratory drive (White et al. 1998; Shea et al. 2; Pillar et al. 21; Akahoshi et al. 21; Malhotra et al. 22). These findings imply that the genioglossus, and possibly other pharyngeal muscles, are activated even on a breath-to-breath basis by physiological negative pressure stimuli and underline the significance of the genioglossus response to NAP in the protection of upper airway patency. We conclude that there is no respiratory modulation of the GGsEMG response to NAP in awake, supine men. It is not clear whether this represents a fundamental property of the mechanism by which upper airway transmural pressure affects upper airway muscle activity in humans, or is attributable to the waking state, posture, or other methodological differences between the present study and prior reports. However, the findings have implications for future studies employing the negative pressure reflex to investigate upper airway muscle activity. References Akahoshi T, White DP, Edwards JK, Beauregard J & Shea SA (21). Phasic mechanoreceptor stimuli can induce phasic activation of upper airway muscles in humans. J Physiol 531,

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