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1Department of Life Science, National Taiwan Normal University, Taipei, Taiwan, Republic of China; and 2Department of Physical Therapy, University of Florida, Gainesville, Florida
Submitted 11 August 2005 ; accepted in final form 30 August 2006
ABSTRACT
Hypoglossal (XII) nerve recordings indicate that pulmonary C-fiber (PCF) receptor activation reduces inspiratory bursting and triggers tonic discharge. We tested three hypotheses related to this observation: 1) PCF receptor activation inhibits inspiratory activity in XII branches innervating both tongue protrudor muscles (medial branch; XIIMED) and retractor muscles (lateral branch; XIILAT); 2) reduced XII neurogram amplitude reflects decreased XII motoneuron discharge rate; and 3) tonic XII activity reflects recruitment of previously silent motoneurons. Phrenic, XIIMED, and XIILAT neurograms were recorded in anesthetized, paralyzed, and ventilated rats. Capsaicin delivered to the jugular vein reduced phrenic bursting at doses of 0.625 and 1.25 µg/kg but augmented bursting at 5 µg/kg. All doses reduced inspiratory amplitude in XIIMED and XIILAT (P < 0.05), and these effects were eliminated following bilateral vagotomy. Single-fiber recordings indicated that capsaicin causes individual XII motoneurons to either decrease discharge rate (n = 101/153) or become silent (n = 39/153). Capsaicin also altered temporal characteristics such that both XIIMED and XIILAT inspiratory burst onset occurred after the phrenic burst (P < 0.05). Increases in tonic discharge after capsaicin were greater in XIIMED vs. XIILAT (P < 0.05); single-fiber recordings indicated that tonic discharge reflected recruitment of previously silent motoneurons. We conclude that PCF receptor activation reduces inspiratory XII motoneuron discharge and transiently attenuates neural drive to both tongue protrudor and retractor muscles. However, tonic discharge appears to be selectively enhanced in tongue protrudor muscles. Accordingly, reductions in upper airway stiffness associated with reduced XII burst amplitude may be offset by enhanced tonic activity in tongue protrudor muscles.
hypoglossal motoneurons; rat
Pulmonary C-fiber (PCF) receptors are unmyelinated vagal afferent neurons innervating the lungs and lower airways. These receptors are stimulated by external irritants such as smoke and ozone (28, 41) and internal chemicals such as histamine, prostaglandin, and bradykinin (28, 42). Activation of PCF receptors can evoke apnea, hypotension, and bradycardia (7, 8, 22, 27, 30, 31). Moreover, neurograms recorded from the main trunk of the XII nerve show that stimulation of PCF receptors via capsaicin injection decreases inspiratory burst amplitude, delays inspiratory burst onset, and triggers tonic discharge (27). Accordingly, PCF receptor activation may result in upper airway narrowing or instability. However, the XII neurograms recorded in the prior study (27) reflect inspiratory neural drive to tongue protrudor and retractor muscles (14) and possibly the intrinsic tongue muscles (2). Prior work suggests that tongue protrudor and retractor muscles respond in parallel to respiratory-related stimuli (18, 33, 39). Accordingly, our first objective was to test the hypothesis that capsaicin-induced PCF receptor activation would result in similar inhibition (reflected as a delay in inspiratory burst onset and decreased burst amplitude) of activity in XII nerve branches innervating tongue protrudor (medial XII nerve; XIIMED) or retractor muscles (lateral XII nerve; XIILAT).
The effect of PCF receptor stimulation on XII motoneuron discharge rate and recruitment is unknown. Decreased XII inspiratory burst amplitude following capsaicin (27) could reflect 1) a decrease in the discharge rate of active motoneurons, 2) cessation of bursting in a subpopulation of motoneurons, or 3) a combination of these two mechanisms. In addition, it is unknown if tonic XII discharge triggered by PCF receptor stimulation reflects de novo recruitment of a subset of XII motoneurons or a change in the discharge pattern of active motoneurons. Thus our second goal was to characterize XII motoneuron activity during and following PCF receptor stimulation via capsaicin. Using a "single-fiber" recording approach (24), we tested the hypotheses that reduced XII neurogram amplitude reflects a decrease in XII motoneuron discharge rate, and tonic discharge reflects recruitment of previously silent motoneurons.
METHODS
Sixty-six male Wistar rats weighing 363 ± 4 g were studied. Twenty-one rats were used to record XIIMED and XIILAT neurograms, and 45 animals were used for recording of individual XII motoneuron action potentials (i.e., single-fiber recordings; 24). All experiments were approved by the Animal Care and Use Committee of National Taiwan Normal University.
General Experimental Preparation
Rats were initially treated with atropine (0.5 mg/kg im, Sigma, St. Louis, MO) and then anesthetized with urethane (1.20 g/kg ip, Sigma). No surgical operation was performed until animals were unresponsive to deep pressure applied to the paws or tail with a hemostat. Supplemental doses of urethane (0.12 g/kg iv) were given as needed to maintain a surgical plane of anesthesia. Rectal temperature was monitored and maintained at 37 ± 1°C with an electric blanket and a heating lamp. Animals remained in a supine position throughout the experiment. The trachea was cannulated caudal to the larynx to enable mechanical ventilation. Catheters were inserted into both the femoral vein and artery for drug administration and monitoring arterial blood pressure (ABP), respectively. Blood pressure was measured by connecting the arterial catheter to a pressure transducer and amplifier (Grass, 7P1, Quincy, MA). A third catheter was inserted into the right jugular vein and placed near the entrance of the right atrium for capsaicin administration, as previously described (27, 30, 31). Subsequently, animals were then paralyzed with gallamine triethiodide (5 mg/kg iv, Sigma) and mechanically ventilated with oxygen. The end-tidal fractional concentration of CO2 (FET
) was continuously monitored by a CO2 analyzer (Electrochemistry CD3A, Ametek, Pittsburgh, PA) connected to the tracheal cannulas via a small length of PE tubing (no. 20) and a needle (27 gauge). The FET was held constant during the experiment (4–5%) by adjusting the frequency (60–70 breaths/min) and tidal volume (3–5 ml/breath) of the ventilator as necessary. A positive end-expired pressure of 3 cmH2O was maintained throughout the experiment to prevent atelectasis. At the end of the experiment, animals were euthanized with an excess dose of urethane (0.6 g/kg iv).
Phrenic and XII Nerve Isolation
The phrenic nerve was identified in the cervical region via a ventrolateral approach and cut as distally as possible (27). The XII nerve branches (i.e., XIIMED and XIILAT) were then exposed with a ventral approach. XIIMED supplies the geniohyoid, the GG, and the verticalis and transversus muscles that comprise the body of the tongue (34). XIIMED was placed over bipolar stainless steel recording electrodes proximal to the projection of fibers to the GG and distal to the geniohyoid branch. Thus XIIMED recordings in our study represented neural drive to the extrinsic and intrinsic tongue protrudor muscles. XIILAT has distinct projections to the extrinsic and intrinsic tongue retractor muscles (SG, HG, and superior and inferior longitudinalis muscles) (34). The HG acts to depress and retract the tongue, whereas the SG acts to elevate and retract the tongue. The XIILAT branch innervating the SG projects from the XII nerve approximately 0–1 mm distal to the primary bifurcation into medial and lateral branches (34). The HG branch projects from XIILAT 
2 mm distal to the XII bifurcation (34). XIILAT was placed over bipolar stainless steel recording electrodes 1 mm distal to the bifurcation of the main XII nerve trunk into medial and lateral branches. Thus XIILAT neurograms represented neural drive to the HG and intrinsic muscles (superior and inferior longitudinalis). Based on the anatomy published by McClung and Goldberg (34), it is unlikely that the XIILAT neurograms reflected neural drive to the SG.
Isolation of Fasciculi Within XIIMED and XIILAT
The XII nerve branches were isolated as described above and then placed in a small tray filled with paraffin oil. The nerves were stripped of connective tissue and then separated into small filaments with a no. 5 forceps and surgical scissors with the aid of a surgical microscope (Leiz). Action potentials from individual motoneurons could be clearly identified based on the shape and amplitude of the waveforms (24, see Fig. 5).
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Phrenic and XII neurograms and XII motoneuron action potentials (see above) were recorded via bipolar electrodes and amplified using an AC preamplifier (Grass, P511, Quincy, MA), filtered (0.33 kHz), and displayed on an oscilloscope (Tektronix 5111, Beaverton, OR). For whole nerve recordings, the signal was also integrated (time constant = 50 ms), and the bipolar electrodes consisted of two stainless steel needles (28 gauge, length = 4 in.) glued together with an interelectrode distance of 1–1.5 mm. For recording of motoneuron potentials, a platinum bipolar electrode was used (diameter = 0.3 mm, length = 3 in.). Data were stored on a hard disk using a commercially available data-acquisitions package (PowerLab, ADI Instruments, Sydney, Australia).
Experimental Protocols
Protocol 1. We recorded phrenic, XIIMED, and XIILAT neurograms prior to and following capsaicin, as follows. The experiment was not initiated until stable inspiratory bursting was observed in all neurograms. Once stable baseline recordings were obtained, three successive doses of capsaicin (0.625, 1.25, and 5 µg/kg) or vehicle solution (see Chemical Preparation) were then infused into the right atrium using a Hamilton microsyringe connected to the right jugular vein catheter. An interval of at least 40 min was allowed between each dose of capsaicin to avoid tachyphylaxis (27). These capsaicin doses were previously established to induce a significant decrease in activity recorded from the main trunk of the XII nerve (27, 30, 31). Twenty-one animals with intact vagus were used for the study of whole nerve response during experiments. Seven of these 21 rats were further studied after bilateral vagotomy.
Protocol 2. We recorded individual motoneuron potentials from axons in XIIMED or XIILAT. After stable motoneuron bursting was confirmed, capsaicin or vehicle was delivered as described in protocol 1. To avoid tachyphylaxis caused by repeated administration of capsaicin, each animal received no more than six administrations of capsaicin. The low, moderate, and high doses of capsaicin induced similar responses in the XII neurograms, and these were not substantially different from the high dose (see Fig. 2). Accordingly, XII motoneuron discharge was examined in response to only the low and moderate doses of capsaicin herein.
Chemical Preparation
Capsaicin (5 mg; Tocris, Bristol, UK) was dissolved in 95% alcohol (1 ml) and polyoxyethylene sorbitan monooleate (Tween 80, 1 ml) as previously described (31). This solution was then diluted with saline (pH 7.4) to obtain a concentration of 500 µg/ml of stock capsaicin. This stock solution was further diluted with saline according to each individual rat's body weight on the day of the experiment and enabled injection volume to be standardized at 5, 10, and 40 µl for 0.625, 1.25, and 5 µg/kg, respectively. Capsaicin was first injected into the right jugular catheter and then infused into the right atrium by injecting 0.3 ml saline. The vehicle was a solution containing 1 ml of 95% alcohol, 1 ml of Tween 80, and 8 ml of saline.
Data and Statistical Analyses
Data stored on the hard disk were retrieved and analyzed by software written with visual C++ in our laboratory. To establish control (i.e., "baseline") neural activity, phrenic, XIIMED, and XIILAT activities were averaged over the 20 respiratory cycles immediately preceding capsaicin administration. For each neurogram, the peak height of the inspiratory burst was calculated by taking the difference of the peak and the end-expiratory baseline value. Inspiratory time (TI) was defined as the total duration of the phrenic inspiratory burst. Expiratory time (TE) was defined as the period between successive phrenic bursts. The total duration of a respiratory cycle was calculated as the sum of TI and TE.
Following capsaicin delivery, the first 15 respiratory cycles were analyzed individually. Neurograms were also analyzed at 1, 2, and 3 min post-capsaicin. Post-capsaicin neurogram activity (peak inspiratory, and tonic) was expressed as a percentage of control. Since tonic XII discharge was often undetectable before capsaicin, the peak tonic discharge was expressed relative to inspiratory activity. The apneic period following capsaicin was defined as the interval between subsequent phrenic discharges; this value was converted into a percentage of the mean TE before capsaicin administration. Phrenic burst duration (TI) was automatically determined by the software program; XII discharge duration was obtained by visually determining the burst onset, and the end point of XII bursting was considered to be the same as the end of the phrenic burst. The differences between phrenic and XII burst duration thus represented the difference in XII discharge onset relative to the onset of phrenic bursting. The mean ABP and heart rate were determined using the data pad module of the PowerLab system.
Individual motoneurons recorded from XIIMED or XIILAT were classified as inspiratory (I) if discharge was observed only during TI, or expiratory-inspiratory (EI) if discharge began some time during TE through TI. Motoneurons that were inactive at baseline but were recruited after capsaicin administration were classified as silent (S). The influence of capsaicin on XII motoneuron discharge rate and onset time was calculated during the first 10 neural breaths during recovery from apnea.
Data were analyzed using multiple comparisons test. Hence, repeated measure of one-way or two-way ANOVA (49) was performed to evaluate significant difference between the control and values post-capsaicin and then followed by the Student-Newman-Keuls post hoc test (SigmaStat 2.0). The impact of vagotomy on phrenic and XII discharge was evaluated by Student's t-test. For all statistical analyses, a P value of <0.05 was considered significant. Data are expressed as means ± SE.
RESULTS
Cardiovascular Responses
Capsaicin evoked cardiovascular responses similar to previous reports (27, 30, 31). ABP dropped by 19 ± 2 and 18 ± 2 mmHg after the low (0.625 µg/kg) and moderate (1.25 µg/kg) capsaicin doses (both P < 0.01, Table 1). Heart rate decreased by 60 ± 6 and 68 ± 5 beats/min following the low and moderate doses of capsaicin (both P < 0.01, Table 1). The high dose of capsaicin (5 µg/kg) caused a biphasic response in ABP (Fig. 1). Mean ABP initially dropped by 15 ± 3 mmHg but then rose by 34 ± 4 mmHg (both P < 0.01 vs. control, Table 1). The initial hypotension was accompanied by a drop of 74 ± 8 beats/min in heart rate (P < 0.01, Table 1). As blood pressure rose, the heart rate also increased but remained 19 ± 3 beats/min below control (P < 0.01, Table 1). After bilateral vagotomy, ABP was 112 ± 4 mmHg (P > 0.05 vs. prevagotomy) and was increased by 9 ± 4 and 18 ± 4 mmHg following the low and moderate doses of capsaicin (P < 0.05 postvagotomy control condition). Control heart rate was not altered by vagotomy (P > 0.05, Table 1). Following low- and moderate-dose capsaicin, heart rate decreased by 29 ± 4 and 34 ± 10 beats/min, respectively, from the postvagotomy control value (both P < 0.01, Table 1).
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Representative examples of phrenic and XII neurogram responses to capsaicin are provided in Fig. 1. Phrenic burst amplitude was transiently reduced following both the low and moderate doses of capsaicin (0.625 and 1.25 µg/kg; P < 0.05, Fig. 2A). Following capsaicin, phrenic burst amplitude returned to control values gradually. With the high dose of capsaicin (5 µg/kg), there was a biphasic response: phrenic burst amplitude decreased for one neural breath (P < 0.05, Fig. 2A) and then enhanced progressively and was statistically greater than control between the 10th and 15th breaths postapnea (P < 0.05, Fig. 2A). Following bilateral vagotomy, phrenic burst amplitude was significantly augmented during the control condition (P < 0.05, Fig. 2A). Capsaicin caused a further increase in phrenic amplitude after vagotomy, but this only achieved statistical significance for breath 10 (Fig. 2A). Vehicle caused no change in phrenic burst amplitude (Fig. 1D).
All three doses of capsaicin resulted in a similar magnitude reduction of both XIIMED and XIILAT inspiratory burst amplitude (Fig. 1, A–C). XIIMED and XIILAT burst amplitude remained below control for the 15 respiratory cycles following capsaicin (P < 0.05, Fig. 2, B and C). However, by 2 min post-capsaicin, XIIMED and XIILAT activity had returned to control, regardless of the capsaicin dose (Fig. 2, B and C). Following bilateral vagotomy (Fig. 1, E and F), XIIMED and XIILAT inspiratory burst amplitude was significantly enhanced (P < 0.01, Fig. 2, B and C) and showed no response to capsaicin (P > 0.05, Fig. 2, B and C). Vehicle did not induce any changes in XIIMED and XIILAT inspiratory burst (Fig. 1D).
Phrenic and XII Neurogram Tonic Discharge
Tonic discharge evoked by capsaicin could be clearly observed by an upward deflection of the integrated neurogram baseline (e.g., Fig. 1, B and C). Moderate and high doses of capsaicin caused mild tonic bursting in the phrenic nerve in approximately one-third of all rats (23/66). However, tonic bursting was always observed in XIIMED and XIILAT recordings after capsaicin (e.g., Fig. 1, B and C). In sharp contrast to the parallel changes in burst amplitude (e.g., Fig. 2), capsaicin caused a significant increase in XIIMED tonic discharge (P < 0.01 vs. control), which was substantially greater compared with XIILAT (P < 0.05, Fig. 3). After bilateral vagotomy, the increase in XIIMED tonic discharge caused by capsaicin was not significant compared with control and was much less than the prevagotomy condition (P < 0.05, Fig. 3). Vehicle and low-dose capsaicin did not induce tonic bursting under any condition (Fig. 1, D and E).
Temporal Characteristics of Inspiratory Bursting
Before bilateral vagotomy, phrenic burst frequency was 62 ± 1.5 cycles/min with a TI of 0.29 ± 0.01 s and TE of 0.70 ± 0.03 s. The TI values of the first two neural breaths following the low and moderate doses of capsaicin were substantially reduced (P < 0.01), but subsequent TI values were not significantly altered (P > 0.05). The low dose of capsaicin (0.625 µg/kg) produced a significant TE prolongation to 213 ± 26% of control (P < 0.05), and the moderate (1.25 µg/kg) and high doses (5 µg/kg) resulted in TE prolongation to 355 ± 49 and 646 ± 68% of control (both P < 0.01).
Representative examples depicting changes in phrenic and XII inspiratory burst onset following capsaicin are provided in Fig. 4. During the control condition, inspiratory burst onset in XIIMED and XIILAT was similar (Fig. 4, Aa and Ba) and always preceded the onset of the phrenic burst (Fig. 4, Aa and Ba). Specifically, the XIIMED and XIILAT bursts occurred at –87.8 ± 8.2 and –79.0 ± 8.2 ms before the phrenic burst, respectively (P < 0.05, Fig. 4, C and D). In response to all three doses of capsaicin, the onset of XIIMED and XIILAT inspiratory activity was delayed such that it began after the phrenic burst (Fig. 4, Ab, Ac, Bb, and Bc). Mean values for this delay were significant (P < 0.05, Fig. 4, C and D). This delay in burst onset was similar between XIIMED and XIILAT neurograms (P > 0.05). The robust tonic discharge in XIIMED made it difficult in some cases to pinpoint the onset of inspiratory bursting (e.g., Fig. 4Ab). However, the single-neuron recordings (see below) confirm the delay in XIIMED inspiratory burst onset following capsaicin.
Following bilateral vagotomy, phrenic burst frequency was reduced to 48 ± 2.7 cycles/min (P < 0.01 vs. prevagotomy). The decreased frequency reflected a prolongation of TE (0.98 ± 0.06 s; P < 0.01 vs. prevagotomy); TI tended to be prolonged, but this did not reach statistical significance (0.30 ± 0.03 s; P > 0.05 vs. prevagotomy). Vagotomy eliminated the effects of capsaicin on TE and frequency (all P > 0.05). Finally, the onset of the XIIMED and XIILAT inspiratory preceded the phrenic burst by a larger degree after vagotomy (Fig. 4, Ad and Bd). Specifically, XIIMED and XIILAT bursting occurred at –225.4 ± 24.6 and –243.3 ± 19.5 ms before the phrenic burst (both P < 0.01, Fig. 4, C and D), respectively, and this relationship was not affected by subsequent capsaicin administration (P > 0.05, Fig. 4, C and D).
XII Motoneuron Discharge
Action potentials were recorded from 153 motoneurons (n = 95 from XIIMED, n = 58 from XIILAT). Representative examples of EI and I motoneuron discharge are shown in Fig. 5; type S neurons are shown in Fig. 7A.
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Mean discharge rate and onset time were quantitatively performed on those neurons receiving the challenge of both the low and moderate doses of capsaicin. During the control condition, XIIMED EI neurons discharged at 19.6 ± 2.3 Hz. The low dose of capsaicin caused a reduction in XIIMED EI discharge to 9.0 ± 3.3 Hz, and the moderate dose caused a similar reduction to 7.0 ± 2.7 Hz (both P < 0.01, Fig. 6, top). EI neurons recorded in XIILAT burst at 18.5 ± 2.8 Hz during control, and after low and moderate doses of capsaicin this was reduced to 11.6 ± 1.6 and 10.8 ± 1.8 Hz, respectively (P < 0.01, Fig. 6, top).
During control conditions, the onset of EI motoneuron bursting in XIIMED occurred at –98.3 ± 34.2 ms before the inspiratory onset of the phrenic neurogram. However, capsaicin altered the burst onset such that XIIMED EI neurons began firing 81.3 ± 39.4 ms (low dose) and 103 ± 36.1 ms (moderate dose) after phrenic burst onset (P < 0.01, Fig. 6, bottom). Similar data were obtained for XIILAT EI neurons. These cells began bursting at –51.3 ± 6.2 ms before the phrenic neurogram burst during control conditions. After capsaicin XIILAT EI discharge began 76.9 ± 18.0 ms (low dose) and 82.8 ± 11.3 ms (high dose) after the phrenic burst (P < 0.01, Fig. 6, bottom). Following vagotomy, two EI neurons were recorded from XIIMED, and one was recorded from XIILAT. These neurons all showed tonic bursting in response to capsaicin injection (data not shown).
I motoneurons.
Inspiratory XII motoneurons quickly reached peak discharge rate and fired throughout inspiration (Fig. 5, A2a and B2a). Following the post-capsaicin apneic period, the majority of I neurons (45/78, pooled XIIMED and XIILAT data) showed a significant decrease in discharge rate, as well as delayed onset of bursting (e.g., Fig. 5, A2b and B2b). In contrast, 40% (33/78) XII motoneurons were completely inhibited following capsaicin and stopped bursting entirely.
The mean discharge rate of XIIMED I neurons was 35.3 ± 1.3 Hz during control conditions. Following capsaicin, this value was reduced to 7.0 ± 3.5 and 3.7 ± 1.8 Hz with the low and moderate doses, respectively (Fig. 6, top). On average, the onset of XIIMED I neuron bursting occurred 22.9 ± 4.4 ms after phrenic burst (Fig. 6, bottom), suggesting that EI neurons (see above) mediate the early onset of the XII neurogram burst. The onset of XIIMED I neuron bursting was further delayed to 136.0 ± 25.6 and 196.0 ± 8.5 ms after the low- and moderate-dose capsaicin, respectively (P < 0.01, Fig. 6, bottom). Discharge rate of I neurons recorded in XIILAT was 27.7 ± 3.5 Hz before capsaicin, and this value was reduced to 2.1 ± 1.2 and 1.8 ± 0.8 Hz after the low and moderate doses of capsaicin (P < 0.01, Fig. 6, top). The commencement of I XIILAT bursting occurred 36.7 ± 5.6 ms after the integrated phrenic burst, and this was further delayed to 96.0 ± 15.0 and 225.0 ± 13.2 ms following the low and moderate doses of capsaicin, respectively (P < 0.01, Fig. 6, bottom).
S motoneurons. Examples of S motoneuron discharge are shown in Fig. 7. Thirteen of 153 XII motoneurons (pooled XIIMED and XIILAT data) were classified as S motoneurons as they were not active during control conditions but were stimulated by capsaicin. Type S motoneurons were observed more frequently in XIIMED (vs. XIILAT) recordings. Specifically, XIIMED recordings revealed 11/95 (12%) S motoneurons (Fig. 7A), and XIILAT recordings showed 2/58 (3%) S neurons (not shown). Once excited, S motoneurons displayed a ramplike increase in discharge, reaching a peak rate of 53.8 ± 8.3 Hz for XIIMED recordings (Fig. 7B) and of 20.0 ± 16.0 Hz for XIILAT recordings. After reaching peak discharge, type S neurons gradually decreased burst frequency until reaching a silent state (Fig. 7, A and B). A few type-S motoneurons were studied before and after vagotomy (XIIMED n = 4, XIILAT n = 2). A variable pattern was observed among XIIMED S neurons after vagotomy: one could no longer be recruited, two neurons were activated in a manner similar to the prevagotomy condition, and one neuron was revealed only after vagotomy (data not shown). Of the two S motoneurons recorded from XIILAT after vagotomy, one responded in a similar fashion to the prevagotomy condition; the other neuron could be only excited by capsaicin before vagotomy (data not shown).
DISCUSSION
There are two major findings of our study. First, capsaicin evoked similar decreases of inspiratory burst amplitude recorded in XII nerve branches innervating tongue protrudor (XIIMED) and retractor (XIILAT) muscles. The diminished burst amplitude primarily reflected a decrease in the discharge rate of both EI and I XII motoneurons. The attenuation of phasic XII bursting by capsaicin was abolished after bilateral vagotomy, indicating that this reflex response is mediated through vagal afferents. Second, capsaicin caused a divergent response in tonic discharge in XIIMED vs. XIILAT. Specifically, the increase in XIIMED tonic discharge was significant compared with control and was more than twofold greater than occurred in XIILAT. This tonic discharge occurred mainly due to recruitment of previously silent XII motoneurons. We speculate that the increase in tonic discharge in XIIMED may act to dilate the pharyngeal airway and thus counteract the increased airway collapsibility associated with diminished XII inspiratory bursting.
Interpretation of XII Nerve Recordings
Although we focus our discussion on the extrinsic tongue muscles, the XIIMED and XIILAT nerve branches also contain motor axons controlling the intrinsic muscles comprising the body of the tongue. Recent studies indicate that the intrinsic tongue muscles are also active during breathing, particularly during states of elevated respiratory drive (2, 3). Thus the XII neurograms and action potentials recorded in the present study may to some degree reflect inspiratory neural drive to the intrinsic tongue muscles. However, the interpretation of the current data is focused on the extrinsic (vs. intrinsic) tongue muscles, primarily because their mechanical actions and functional contribution to breathing are better defined (14). Nevertheless, intrinsic tongue muscle contraction may make a significant contribution to the regulation of pharyngeal airway patency during breathing (2, 3), and intrinsic tongue muscle activity may be altered by PCF activation.
Another potential concern is the interpretation of tonic XII discharge. Tonic activity was minimal before capsaicin, and accordingly it was problematic to quantify post-capsaicin activity relative to the baseline condition. Accordingly, tonic XII neurogram activity was expressed relative to the peak inspiratory activity. This normalization approach may in fact overestimate the relative tonic activity due to strong inhibition of XII bursting caused by lung inflation. However, the single-motoneuron recordings confirm the affect of capsaicin on tonic XII discharge and show that this response appears to result from reflex recruitment of previously silent XII motoneurons.
Interpretation of Capsaicin Effects
Capsaicin delivery into the right atrium has previously been used to activate PCF receptors and evoke cardiopulmonary reflexes, and it is generally accepted that PCF receptor activation causes apnea (8–10). However, in addition to PCF receptor-mediated responses, capsaicin may also influence respiratory motor output via excitation of A
-fibers or rapidly adapting receptors (5, 22, 40). Excitation of vagal A-fibers can provoke augmented breaths and tonic diaphragm activity (35, 40). Accordingly, the biphasic phrenic response (i.e., immediate decrease followed by a delayed increase) observed after high-dose capsaicin may indicate that A-fibers were stimulated. In contrast, high-dose capsaicin decreased XII neurogram inspiratory burst amplitude, and capsaicin also inhibited XII motoneuron discharge. Thus A-fibers, if stimulated by capsaicin, did not appear to trigger increases in respiratory-related XII discharge. In any case, the effects of capsaicin on respiratory-related XII bursting were clearly vagally mediated, as they were abolished by bilateral vagotomy.
As indicated above, activation of vagal A-fibers can initiate tonic diaphragm activity (35, 40). This prompts us to consider whether A-fiber stimulation by capsaicin may also cause tonic XII bursting. For example, the high dose of capsaicin that triggered both enhanced phrenic bursting and reduced XII burst amplitude also concomitantly evoked tonic XII bursting. This observation raises the possibility that PCF receptor activation can inhibit XII bursting, while simultaneous A-fiber stimulation may trigger tonic XII discharge. This view is supported by the biphasic response of blood pressure and phrenic burst amplitude to high-dose capsaicin in the present study. Specifically, it has previously been reported that activation of PCF receptors and A-fibers produces hypo- and hypertension, respectively (40). However, it is likely that extravagal afferents contributed to the cardiovascular reflexes because hypertensive response was still observable with capsaicin administration after bilateral vagotomy. In addition, the extravagal afferents may also contribute to the reflex excitation of tonic XII discharge. For example, after vagotomy, capsaicin induced a mild tonic XIIMED discharge that was not statistically significant, and XII motoneuron recruitment was attenuated.
In these experiments, respiratory discharge in the vagally intact animals was entrained with the rate of the mechanical ventilator as previously described (36, 44). However, the volume and cycle of the ventilator were adjusted and maintained at constant levels over the entire experiment; thus the relative degree of entrainment was similar for each animal and across the control and capsaicin conditions. Accordingly, any changes in phrenic and XII bursting should primarily reflect capsaicin administration. Nevertheless, we cannot discount a potential interaction between ventilator entrainment and capsaicin-induced respiratory responses.
Respiratory-Related Control of Extrinsic Tongue Muscles
In humans, inspiratory GG EMG activity is minimal during eupneic breathing (26, 33, 50), but the GG is recruited when respiratory drive is increased by exercise or hypoxia (26, 50). Tongue retractor muscles are also quiescent during eupnea in humans but are recruited in parallel with the GG during chemoreceptor stimulation (33). Inspiratory coactivation of tongue protrudor and retractor muscles also occurs during quiet breathing and chemoreceptor stimulation in animals (17, 18, 37), and XII burst onset normally occurs before onset of phrenic inspiratory bursting (15, 16, 25, 27, 37, 46, 47). In addition, inspiratory activity in both tongue protrudor and retractor muscles is similarly inhibited by phasic lung volume feedback (4) and augmented by upper airway negative pressure (39). Our data extend these observations by showing that the inhibitory effects of capsaicin-induced PCF receptor activation are similar for both tongue protrudor and retractor muscles. Inhibition of XII neurogram output was manifest as both reduced burst amplitude and delayed burst onset. Consistent with these observations, both EI and I motoneurons recorded in XIIMED and XIILAT showed decreased discharge rate and delayed burst onset after capsaicin. Importantly, inspiratory-related neurogram and motoneuron activity recorded in XIIMED and XIILAT responded similarly, confirming prior reports of parallel respiratory modulation of tongue protrudor and retractor muscles (4, 17, 18, 37, 39).
Our results suggest at least two possibilities for PCF receptor-mediated control of XII motoneurons. First, afferent signals from vagal C-fibers may be evenly distributed on inspiratory XII motoneurons controlling tongue protrudor and retractor muscles. However, a second possibility is that afferents of vagal C-fibers activated by capsaicin converge on a common excitatory premotor population projecting to inspiratory XII motoneurons with axons in XIIMED and XIILAT (37). If so, signals from vagal afferents may inhibit this excitatory premotor population, with a net result of parallel inhibition of XIIMED and XIILAT inspiratory motor output.
Tonic XII Activity
Haxhiu et al. (21) previously reported that following capsaicin, the duration of electrical silence is longer for the diaphragm than for the GG (21). Our data seem to be consistent with this report as tonic bursting in XIIMED occurred before phasic phrenic bursting during recovery from apnea. However, the physiological meaning of phasic vs. tonic activity may differ. For example, if upper airway patency is compromised by the decreased and delayed onset of phasic XII bursting, this affect may be ameliorated by the elevated tonic discharge of the XIIMED branch in the face of the capsaicin challenge or other irritant. Our data suggest that previously silent XII motoneurons are recruited to produce tonic activity in the GG muscle.
Our data do not allow definitive conclusions regarding the physiological mechanisms that trigger tonic discharge and the selective increase in tonic XIIMED (vs. XIILAT) activity. This response could be triggered by the transient hypotension that occurred following capsaicin (e.g., Fig. 1, B and C). However, it was previously noted that hypotension is associated with similar changes in tonic discharge in both XIIMED and XIILAT branches (17). Accordingly, we suggest that the divergent tonic XII discharge patterns reported here resulted primarily from capsaicin-induced activation of vagal afferents. Ryan and colleagues (39) reported that airway pressures of –20 to –30 cmH2O evoked a more robust increase in tonic XIIMED than in XIILAT. Thus the divergent tonic XII discharge patterns reported here are not uniquely evoked by vagal afferent activation and may promote airway stability under a range of physiological conditions.
The neuroanatomic substrate underlying the divergent effects of vagal afferent activation on tonic XII activity is unclear. Segregation of some portion of the neural networks controlling tongue motoneurons is expected because many tongue movements require selective activation of tongue protrudor or retractor muscles. Consistent with this suggestion, Dobbins and Feldman (12) demonstrated that protrudor and retractor motoneurons have both differential and common premotor synaptic inputs. Our data suggest that vagal afferent activation may selectively stimulate tonic inputs to tongue protrudor (vs. retrusor) motoneurons. Consistent with this suggestion, the type-S motoneurons, which are apparently responsible for tonic discharge following capsaicin, were observed more frequently in XIIMED than XIILAT recordings.
Physiological Significance
Our data suggest that PCF receptors influence XII motor output in two important ways. First, PCF receptor stimulation results in an uncoupling of inspiratory related phrenic and XII motor outputs such that XII activity is delayed relative to the phrenic burst. The normally occurring "preinspiratory" XII discharge has been proposed to stabilize the pharyngeal airway before the drop in airway pressure induced by diaphragm contraction (23, 25, 38, 45). Second, PCF receptor stimulation results in a significant reduction in XIIMED and XIILAT burst amplitudes that persists for several breaths after capsaicin administration. The coactivation pattern of XIIMED and XIILAT bursting has previously been shown to reduce upper airway collapsibility (1, 19). Thus both reduced XIIMED and XIILAT amplitude and burst onset may have significant consequences for pharyngeal airway patency in the face of transmural pressures or irritant stimulations favoring upper airway narrowing or collapse. However, these "negative" changes in XII motor output following capsaicin may be offset, to some degree, by the transient tonic discharge observed in the XIIMED nerve branch. Indeed, previous authors have suggested that tonic GG EMG may promote upper airway XIIMED stability across the respiratory cycle (43). Tonic XIIMED discharge could persist under conditions where vagal afferents are continually activated. Conditions that could evoke continued vagal afferent activation include pulmonary edema (11) and air embolism (7). Under these pathological conditions, activation of vagal afferents would be expected to diminish the inspiratory bursting in both the XIIMED and XIILAT, possibly resulting in upper airway instability. However, a concomitant increase in XIIMED tonic discharge under these conditions could minimize the probability of upper airway narrowing or collapse. Indeed, tonic electrical stimulation of XIIMED results in an increase in inspiratory airflow rates in isolated upper airway preparations (13, 19) and also an increase in pharyngeal airway dilatation (6).
GRANTS
This study was supported by a research grant from the National Science Council (NSC91–2320-B-003–002) and also a research grant from the National Taiwan Normal University (ORD94–3), Taipei, Taiwan, ROC.
ACKNOWLEDGMENTS
We thank Dr. Win-Tai Savio Cheng for reading this manuscript and for advice.
FOOTNOTES
Address for reprint requests and other correspondence: J.-C. Hwang, Dept. of Life Science, National Taiwan Normal Univ., Taipei, Taiwan, ROC (e-mail: jchwang{at}ntnu.edu.tw)
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.
REFERENCES
and C-fibers. Pulm Pharmacol Therap 16: 1–7, 2003.[CrossRef][ISI][Medline]This article has been cited by other articles:
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  K.-Z. Lee, D. D. Fuller, I-J. Lu, L.-C. Ku, and J.-C. Hwang Pulmonary C-fiber receptor activation abolishes uncoupled facial nerve activity from phrenic bursting during positive end-expired pressure in the rat J Appl Physiol, January 1, 2008; 104(1): 119 - 129. [Abstract] [Full Text] [PDF]
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