Prolonged augmentation of respiratory discharge in hypoglossal motoneurons following superior laryngeal nerve stimulation

Brain Research, 538 (1991) 215-225

215

Elsevier BRES 16225

Prolonged augmentation of respiratory discharge in hypoglossal motoneurons following superior laryngeal nerve stimulation Chun Jiang*, Gordon S. Mitchell** and Janusz Lipski Department of Physiology, Universityof Auckland, Auckland (New Zealand) (Accepted 7 August 1990)

Key words: Hypoglossal motoneuron; Intracellular recording; Serotonin; Superior laryngeal nerve; Carotid sinus nerve; Immunohistochemistry; Respiratory control

Experiments were conducted to investigate long-lasting effects of brief superior laryngeal nerve (SLN) stimulation on respiratory discharge in the hypoglossal nerve. In paralyzed, decerebrate and artificially ventilated cats, SLN stimulation (50 Hz, 3-5 s, 3-5 times threshold for inhibition of phrenic nerve discharge) immediately increased hypoglossal activity. Following stimulation, the amplitude of respiratory activity in the hypoglossal nerve was augmented (478 + 205%), and slowly decayed to prestimulus levels with a time constant of 106 + 16 s. In contrast, phrenic nerve activity was completely inhibited during the SLN stimulation and for several seconds thereafter. After activity resumed, phrenic burst frequency remained depressed (33 + 6%). Stimulation of the carotid sinus nerve elicited similar effects on hypoglossal nerve activity. Intracellular recordings from hypoglossal motoneurons indicated that SLN stimulation increased central respiratory drive potentials (CRDPs) following a stimulus train, but had inconsistent effects on resting membrane potential. Intracellular depolarizing current pulses (5-15 nA; 2 s) had no prolonged effects on membrane potential or CRDPs. The possible role of serotonin in prolonged augmentation of hypoglossal activity following SLN stimulation was investigated. Intracellular injection of horseradish peroxidase (HRP) into hypoglossal motoneurons and immunohistochemistry for serotonin revealed some close appositions between serotonin immunoreactive boutons and HRP-labeled neurons, but such appositions were sparse. Pretreatment with methysergide had little effect on prolonged augmentation of hypoglossal discharge following SLN stimulation. These results indicate that: (1) SLN stimulation causes prolonged augmentation of hypoglossal activity probably via increased synaptic inputs to hypoglossal motoneurons; and (2) serotonin is not necessary in the underlying mechanism. INTRODUCTION The ventilatory response to a number of peripheral inputs persists beyond the period of afferent stimulation (cf. ref. 10). Such prolonged effects can be considered a form of respiratory memory, and occur in several time domains ranging from seconds to hours. For example, electrical stimulation of the carotid sinus nerve (CSN) immediately increases phrenic discharge, but the augmentation only slowly decays to control levels after stimulation is terminated. The decay of phrenic discharge to control levels reflects two distinct processes with time domains of minutes (afterdischarge) to hours (long term facilitation) (cf. ref. 10). In contrast with long term facilitation, afterdischarge is of shorter duration, is elicited by a wider range of afferent inputs, does not require monoamine neurotransmitters, and is suspected to result either from 'reverberating' neuronal circuits (cf. ref. 10) or a mechanism analogous to short term potentiation 42. Conversely, activation of afferent fibers in the superior laryngeal nerve (SLN) inhibits phrenic

activity, and this inhibition outlasts the duration of stimulation (cf. refs. 10, 18). Most documented examples of prolonged respiratory effects concern phrenic nerve activity or overall ventilation. As yet, there are few examples of m e m o r y in other respiratory motoneuron pools. In the last decade or so, there has been a great deal of interest in the control of upper airway muscles and their supportive role in respiration (cf. refs. 3, 18, 31). Much of this interest stems from the recognition that upper airway muscles are essential in maintaining upper airway patency, offsetting the tendency of the flaccid upper airways to collapse due to negative pressures created by thoracic muscles (e.g. ref. 29). For example, pharyngeal occlusion may result whenever the force of negative intrapharyngeal pressure is greater t h a n the opposing force generated by contraction of the genioglossus muscle 4,3o. Inspiratory activity of the genioglossus is strongly activated by stimulation of afferent fibers in both SLN and CSN (cf. refs. 18, 31). Therefore, both afferent

* Present address: Department of Pediatrics, School of Medicine, Yale University, New Haven, CT 06520, U.S.A. ** Present address: Department of Comparative Biosciences, University of Wisconsin, Madison, WI 53706, U.S.A. Correspondence: J. Lipski, Department of Physiology, School of Medicine, University of Auckland, Private Bag, Auckland, New Zealand. 0006-8993/91/$03.50 t~) 1991 Elsevier Science Publishers B.V. (Biomedical Division)

216 pathways are likely to play an important role in maintaining upper airway patency during conditions such as sleep or anesthesia when upper airway tone is diminished (cf. refs. 18, 31). It is of considerable interest to determine if brief activation of SLN or CSN afferent fibers also exerts prolonged effects on respiratory discharge in hypoglossal motoneurons since prolonged activation of the genioglossus would help preserve upper airway patency for longer periods following upper airway collapse. Increased discharge of motoneurons following afferent stimulation could result in two ways: (1) they could receive greater synaptic inputs that persist following the period of stimulation; and/or (2) they could become more excitable, causing a greater response to the same synaptic inputs. Examples of augmented motoneuron discharge following brief activation can be found for both alternatives. Both afterdischarge and long term facilitation of phrenic motoneurons following CSN stimulation have been suggested to result from a prolonged increase in synaptic inputs (cf. ref. 10). In contrast, lumbar motoneurons exhibit long lasting plateau potentials following either synaptic activation or direct depolarization, leading to greater excitability5"6'15. Notably, both long term facilitation of phrenic motoneurons following CSN stimulation 2s'26 and plateau potentials in lumbar motoneurons ~5 require the presence of monoamines, particularly serotonin. Serotonin containing nerve terminals can be found in the hypoglossal nucleus 1. In addition, some patients with obstructive sleep apnea have been successfully treated with drugs that block reuptake of serotonin (cf. ref. 14), suggesting that serotonin may indeed be involved in the regulation of upper airway muscle tone. There were 5 primary objectives of this study: (1) to determine if electrical stimulation of the SLN elicits prolonged effects on respiratory related discharge in hypoglossal motoneurons; (2) to determine if prolonged augmentation is associated with greater tonic versus phasic membrane depolarization (using intracellular recording techniques); (3) to determine if serotonin plays a role in the response of hypoglossal motoneurons to SLN stimulation; (4) to compare the effects of SLN and CSN stimulation on hypoglossal motoneuron discharge; and (5) to test the hypothesis that hypoglossal motoneurons express plateau potentials following direct intracellular depolarization.

Cannulas were placed in the trachea and a femoral artery and veto. Both vagi were left intact in most experiments. The C5 branch of the phrenic nerve, as well as the hypoglossal, superior laryngeal and carotid sinus nerves were isolated on the left side and prepared for standard bipolar recording or stimulation. In two experiments, an external and/or internal intercostal nerve (Ts-T~,) was isolated and prepared for recording or stimulation; in one experiment, the left vagus nerve was isolated and prepared for stimulation. The animals were paralyzed with p a n c u r o n i u m bromide (Pavulon, 0.5 mg/kg, i.v. ; supplemented as needed) and artificially ventilated. To improve stability of intracellular recordings, the animals were ventilated at a high respiratory frequency and low tidal wglume. Tidal volume was adjusted until an end-tidal CO 2 fraction between 0.04 and 0.05 was attained at a ventilator frequency of 60-70/rain. Supplemental oxygen was added to the inspiratory gas to maintain arterial oxygen tensions above 100 m m Hg. The dorsal surface of the medulla was exposed by craniotomy and cerebellectomy. Bilateral p n e u m o t h o races were made and an expiratory load of 1-3 cm H 2 0 was applied. Rectal temperature was recorded and servocontrolled near 37-38 °C. Intravenous fluids (lactated Ringer's) were given in an effort to maintain a normal extracellular fluid volume. Blood pressure and tracheal pressure were monitored. Arterial blood samples were periodically drawn anaerobically into heparinized syringes, capped and stored on ice until analyzed for pH, p , C O 2 and p~O 2 (Radiometer, A B L 330). Arterial bicarbonate concentration and the base excess were calculated, and corrections were made for temperature differences between the cat and blood gas analyzer. Sodium bicarbonate was administered (i.v.) as necessary to maintain the arterial bicarbonate concentration above 20 mEq/liter. Signals recorded from the phrenic and hypoglossal nerves (as well as the intercostal nerve in two cats) were amplified, filtered (60 Hz to 3 kHz), full wave rectified and R C integrated (time constant, 50 ms). The amplitude and frequency of integrated bursts were used as an index of respiratory discharge in each m o t o n e u r o n pool; phrenic bursts were used to indicate the central inspiratory phase. Intracellular recordings were made from antidromically identified hypoglossal m o t o n e u r o n s using glass microelectrodes (ca. I ktm tip) filled with either 3 M KCI, 2 M potassium citrate or a 10% solution of horseradish peroxidase (HRP) in 0.05 M Tris-HCI buffer (pH 7.6) containing 0.3 M KCI. lntracellular recordings. After successful penetration of a hypoglossal m o t o n e u r o n , a positive or negative bias current was applied through the recording electrode until the neuron was near the threshold for action potential generation. Brief depolarizing or hyperpolarizing pulses (5, 10 or 15 n A ; 2 s duration) were applied in an effort to detect plateau potentials (cf. ref. 15). In addition, the response to SLN stimulation was determined (3 s; 50 Hz; 0.1 ms duration; 3 times threshold) while efferent activity in the hypoglossal nerve was monitored. Hyperpolarizing pulses (10 or 15 nA; 2 s) were passed through the recording microelectrode in an effort to reverse prolonged augmentation of m o t o n e u r o n discharge caused by SLN stimulation. Neurons were considered in the analysis only if: (1) they were tested through a significant part of this protocol while the m e m b r a n e potential remained below - 4 5 mV, (2) the hypoglossal nerve remained active and responsive to SLN stimulation; and (3) m e a n blood pressure remained above 75 m m Hg. In neurons to be filled with HRP, the response to SLN stimulation was first observed to classify the behavior of the m o t o n e u r o n . H R P was injected iontophoretically using 2 - 8 n A depolarizing current pulses (80% duty cycle; 2 Hz) for 10-20 min.

Experimental protocols Stimulation of superior laryngeal nerve (SLN). To begin each MATERIALS AND METHODS

General procedures and recordings Experiments were conducted on 10 adult cats (1.5-3.5 kg). Nine were decerebrated at the midcollicular level under halothane anesthesia (with decerebellation); one cat was anesthetized with a mixture of chloralose (40 mg/kg) and urethane (250 mg/kg).

protocol, :¢entilation was adjusted until a constant level of arterial p C O 2 (paCO2) was attained that was at or slightly above the threshold for rhythmic (respiratory) discharge in the hypoglossal neurogram. The range of paCO2 values was 32-49 m m Hg with a m e a n of 41 + 2 m m Hg (+ S.E.M.; n = 8). T h e SLN was stimulated while recording phrenic and hypoglossal nerve activity (as well as external intercostal in two cats). Three or 5 s trains of stimuli were

217 used (50 Hz, 0.1 ms pulse duration) at an intensity of 3-5 times the threshold for detectable inhibition of the phrenic neurogram (threshold, 0.1-0.3 V). Stimulus trains were repeated 2-5 times with intervals between trains of 1 or 2 min. Following the last period of SLN stimulation, a second arterial blood sample was drawn to ensure that paCO 2 had not changed during the protocol. In 6 cats, the protocol was repeated 30-50 min following administration of the serotonin receptor antagonist, methysergide (1 or 2 mg/kg, i.v.); this dose blocks both long term facilitation of phrenic discharge following CSN stimulation26 and expression of plateau potentials in lumbar motoneurons6. Since methysergide inhibits ventilatory activity and blood pressure26, p a C O 2 w a s elevated if necessary to assure that P a C O 2 w a s near the threshold for rhythmic discharge in the hypoglossal nerve. Reflex responses to SLN stimulation were analyzed only if: (1) mean arterial blood pressure remained above 75 mm Hg; (2) P a C O 2 did not change by more than 1 mm Hg before vs after stimulation; and (3) hypoglossal nerve activity showed evidence of at least some phasic activity, synchronized with the phrenic neurogram. Values reported are means from prestimulus controls and from the final stimulus train of a series. No evidence was found for cumulative effects with successive stimulus trains. Stimulation of carotid sinus nerve (CSN). Experiments were conducted in 4 cats to compare the effects of CSN stimulation on hypoglossal and phrenic nerve activity. Following an arterial blood sample, the CSN was stimulated for 30 s to 2 min at 3-5 times the threshold for reflex excitation of phrenic activity (25 Hz, 0.5 ms duration, 1-2 V). Stimulation was repeated 3-5 times at intervals of 2-10 min. A second blood gas sample was drawn at the end of the protocol to determine if paCO 2 had remained constant. Only two experiments were conducted following pretreatment with methysergide and the results were ambiguous; therefore, these results are not presented.

Histology and immunohistochemistry At least 1 h after the last neuron was injected with HRP, cats were perfused transcardially with 0.9% NaCl, and then 4% formaldehyde with 0.2% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4). Vibratome sections (75 /~m) were cut in the sagittal plane. The morphology of the HRP-filled cells was revealed with a standard histochemical reaction 13. Sections were washed 3 times in Tris-phosphate buffer containing 0.3% v/v Triton X-100 (pH 7.4) for 10 min. Subsequently, they were incubated in a rabbit antibody to serotonin (Dr. W. Watkins, Auckland) diluted 1:2000 in the same buffer, containing 20% heat-inactivated porcine serum (Sigma) for 48 h at room temperature. Following 3 additional rinses (10 min), the sections were incubated overnight in a biotinylated sheep anti-rabbit antibody (Sigma B9140) in 1% porcine serum. After 3 further washes, the sections were incubated for 1 h in an avidin-peroxidase conjugate (1:1000, Sigma) and washed again. The location of serotonin immunoreactive nerve terminals was revealed by incubating sections in 50 mM Tris-HCl buffer (pH 7.6) containing 0.02% diaminobenzidine, 0.6% nickel ammonium sulphate, and 0.003% hydrogen peroxide. Following washing, the sections were mounted onto gelatin subbed slides, dehydrated, cleared in xylene and mounted with DePeX. Possible appositions of serotonin immunoreactive boutons with HRP labeled neurons were examined using oil immersion and × 100 objective. Boutons and labeled neurons were considered to form close appositions if they were found side by side with no discernible gap between them.

RESULTS

Stimulation o f S L N T h e r e s p o n s e s of p h r e n i c a n d hypoglossal n e r v e activity to S L N s t i m u l a t i o n are illustrated in Fig. 1. A

short stimulus train (5 s, 50 Hz) to the S L N caused a n i n h i b i t i o n of p h r e n i c n e r v e activity, s t r o n g a u g m e n t a t i o n of activity in the hypoglossal n e r v e in b l o o d pressure. P h r e n i c bursts total of 13 + 9 s ( + S . E . M . ; n = 8). r e t u r n e d , burst f r e q u e n c y was 3 3 %

a n d a small increase were i n h i b i t e d for a O n c e p h r e n i c activity ( + 6 % S . E . M . ) lower

d u r i n g the first 5 p o s t s t i m u l a t i o n bursts relative to p r e s t i m u l u s c o n t r o l levels (17.9 + 2 . 7 / m i n control vs 11.1 + 0.9/min p o s t s t i m u l u s ; P < 0.01, p a i r e d t-test); burst f r e q u e n c y slowly r e t u r n e d to p r e s t i m u l u s levels. T h e decrease in f r e q u e n c y f r o m c o n t r o l to the first 5 poststim u l a t i o n p h r e n i c bursts was c o r r e l a t e d with the initial burst f r e q u e n c y of the cats; the g r e a t e r the initial f r e q u e n c y , the g r e a t e r the decrease following SLN s t i m u l a t i o n (slope = - 0 . 7 3 + 0.09; r 2 = 0.93; P < 0.0001, t-test for significant slope). P o s t s t i m u l u s effects of SLN s t i m u l a t i o n o n p h r e n i c burst a m p l i t u d e were small a n d variable. All effects o n e x t e r n a l intercostal n e r v e activity were similar to p h r e n i c ( n o t s h o w n ) . D u r i n g s t i m u l u s trains, tonic hypoglossal n e r v e activity i n c r e a s e d with s u p e r i m p o s e d short periodic bursts; the f r e q u e n c y of these bursts was 1.2 + 0.1 H z (n = 8). A f t e r stimulus trains e n d e d , activity in the hypoglossal n e r v e i m m e d i a t e l y decreased, b u t e x h i b i t e d persistent tonic activity that slowly d e c a y e d t o w a r d s p r e s t i m u l u s c o n t r o l values; the short p e r i o d i c bursts d i s a p p e a r e d i m m e d i a t e l y at the e n d of a stimulus train. A f t e r a p a u s e of 3.2 + 0.4 s (n = 8) following s t i m u l a t i o n , r e s p i r a t o r y discharge in

XII 20S"' Phr

BP

~Jj~Jj~j~J~J~JJ~JjJJjJ, i20mmH~ 70

~_-_-_-_J]__

~-----___

_]IU] . . . . . . __

!JJJJJ~JJ~ JJJJ 11

Fig. 1. Responses of integrated hypoglossal (XII) and phrenic (Phr) neurograms and arterial blood pressure (BP) to repeated superior laryngeal nerve (SLN) stimulation in a decerebrate cat. Horizontal bars under the BP record indicate SLN stimulus trains (50 Hz, 0.1 ms duration, 3 times threshold, 5 s). During each stimulus train, hypoglossal motoneurons were strongly activated, the phrenic nerve was inhibited and blood pressure increased. The amplitude of respiratory modulation in the hypoglossal nerve remained elevated after the stimulus train had ended and slowly returned to prestimulus control levels over a period of several minutes. Phrenic (and hypoglossal) burst frequency was depressed following SLN stimulation; this particular cat had the highest prestimulus frequency of the experimental group, and exhibited the greatest poststimulus slowing (see text). There was no apparent difference in paCO2 before vs after the final stimulus train, indicating that changes in chemical stimuli are unable to account for the poststimulus augmentation of XII.

218 the hypoglossal nerve resumed, but with a slowed rate as described for the phrenic neurogram. On the other hand, the amplitude of hypoglossal inspiratory bursts was augmented by 67 to 1800% during the first poststimulus burst (average prestimulus amplitude/poststimulus amplitude = 0.30 + 0.06; significantly different from 1.0, P < 0.001). The augmentation of hypoglossal burst amplitude decreased approximately as an exponential function of time with a time constant (63%) ranging from 40 to 184 s (mean 106 + 16 s). On 3 occasions, the augmentation could still be observed (although much reduced) 10 rain following SLN stimulation (Fig. 2). The persistent elevation of hypoglossal burst amplitude could not be accounted for by changes in p~CO2 or blood pressure. Qualitatively similar effects were observed in all 10 cats used in this study. On several occasions, an effort was made to reverse the prolonged augmentation of hypoglossal nerve discharge by reflex inhibition via stimulation of the vagus (n = 1) or the internal and external intercostal nerves (n = 2). No evidence was found that the mechanism responsible for prolonged augmentation of hypoglossal discharge could be reset by 5 s inhibitory stimulus trains. Prolonged hypoglossal nerve augmentation was dependent on the level of paCO2 . Its duration was considerably shorter when paCO 2 was lower and prestimulus hypoglossal activity was either very small or absent. During these long experiments, it was apparent that reflex actions of SLN stimulation on hypoglossal nerve activity were not robust and often deteriorated as the experiment progressed. The reasons for this deterioration are not clear and may relate to repeated strong stimulations of the reflex pathway or to general deterioration of the animal preparation. The effects of pretreatment with methysergide (1 or 2 mg/kg) on hypogiossal and phrenic nerve responses to SLN stimulation are shown in Fig. 2. Although rhythmic discharge in the hypoglossal nerve was barely evident prior to SLN stimulation in this example, the effects of SLN stimulation on phrenic and hypoglossal nerve discharge were qualitatively similar to pretreatment effects in the same animal. However, the prolonged increase in hypoglossal activity is smaller than in control conditions, an effect that may be due to the lower level of respiratory drive. In two of the 6 cats, blood pressure decreased markedly and the condition of the animal became very poor following administration of methysergide; responses from these cats were not analyzed. The remaining 4 cats all exhibited qualitative evidence for persistence of prolonged augmentation of hypoglossal nerve discharge following SLN stimulation, suggesting that serotonin is not necessary in the mechanism underlying this phenomenon.

Stimulation of CSN The effects of CSN stimulation for 30 s on phrenic and hypoglossal nerve activities are shown in Fig. 3. During CSN stimulation, phrenic and hypoglossal nerve activities and arterial blood pressure are increased. The relative effect of CSN stimulation on phrenic amplitude is lower during hypercapnia than in normocapnia or hypocapnia (e.g. ref. 9). Thus, the relatively high background level of paCO2, established to assure respiratory hypoglossal discharge, is expected to decrease the increment in phrenic activity (Fig. 3). Following stimulation, phrenic activity returned quickly to control levels. There was no prolonged change in burst frequency following CSN stimulation when comparing prestimulus control levels with the first 5 bursts following the final stimulus train (14.5 + 2.5 control vs 11.6 + 0.5 poststimulus; n = 4; P > 0.1). Hypoglossal nerve activity was strongly activated by CSN stimulation; both tonic and short bursts of activity

PRE-METHYSERGIDE

A

/

E

!

Phr gJ±~J_)~LIJJ2JJJJJJJJjJ.JJJJJJJJ~l?~L~J22JJJJJJ±t

WJJJJJ.~J wJ2JJ±L!

130mmHg BP[

.

.

.

.

.

=_

"

so

i m l l m m mmmum ~

POST-METHYSERGIDE 1mg/k 9

~

2 mg/k g

Xll 30s

Phr

BP

U2tJ~,JdJJJJJJJ~JJ&JOJJJJJJJJJJ,JJJ~JJJJ2JJJ~JJJdtdJ&~jZC ~-~o I" L

........ .

II1

80

Fig. 2. Responses to SLN stimulation before (A) and after (B,C) pretreatment with methysergide in a decerebrate cat. In A, a response similar to that shown in Fig. 1 is shown; however, the prestimulus control activity of the hypoglossal nerve showed only slight respiratory modulation. Horizontal bars indicate periods of SLN stimulation; arrows indicate gaps in the recording of 5 min. Records as long as 10 min following SLN stimulation showed prolonged elevation of rhythmic hypoglossal discharge. In 13 (1 mg/kg) and C (2 mg/kg), pretreatment with methysergide diminished the poststimulus augmentation of hypoglossal discharge, but the same qualitative response is evident. Recordings made approximately 20 min after administration of methysergide. In this cat, we did not increase pCO 2 after methysergide administration since rhythmic discharge could still be heard with an audio monitor. Abbreviations as in Fig. 1. The time scales are similar in A, B and C.

219 were evident. Following the final stimulus train, the amplitude of the first hypoglossal burst was elevated between 100 and 400% (prestimulus amplitude/poststimulus amplitude = 0.39 + 0.10; significantly different from 1.0, P < 0.01; n = 4). The prolonged augmentation of hypoglossal nerve activity decreased approximately as an exponential function of time with a time constant ranging from 58 to 257 s (mean 131 + 63 s). These effects were consistent in all 4 cats tested and were within the range encountered following SLN stimulation.

Intracellular recordings of hypoglossal motoneurons Fifty-eight hypoglossal motoneurons studied intracellularly were considered acceptable based on the selection criteria previously outlined (see Materials and Methods). All neurons but 5 were located between the obex and 1 mm caudal to the obex; the remaining 5 were more caudal. Neurons were observed both with and without central respiratory drive potentials. They were classified according to their responses to SLN stimulation, and a summary is presented in Table I. An example from one important class of neurons (n = 18) is shown in Fig. 4. These neurons were strongly depolarized during stimulation of the SLN and typically exhibited central respiratory drive potentials (CRDPs) prior to SLN stimulation; at the end of a stimulus train, a long lasting augmentation of the CRDPs (n = 15) and/or brief tonic depolarization (n = 3) could be observed. The example shown in Fig. 4 exhibits both increased C R D P amplitude and tonic depolarization.

TABLE I

Classifications of hypoglossal motoneurons studied via intracellular recordings Response to S L N stimulation

n

CR D Ps

Tonic activity only

(+)pa (+)na (-) Not tested

18 11 16 13

15 5 6 7

3 6 10 6

Total

58

33

25

CRDPs, neurons that exhibit central respiratory drive potentials; n, total number of neurons in each classification; SLN, superior laryngeal nerve; (+) pa, neurons depolarized during stimulation with post-stimulus augmentation; (+) na, neurons depolarized during stimulation with no detectable post-stimulus augmentation; (-), neurons without response during or after stimulation.

However, the latter effect was of short duration relative to the effects on C R D P amplitude. We were unable to

A

MP-i

'0~

-70

!: .:

,

120

aP

[

. . . . . . ~iiiii~3 2=' ..............i

70mmHg

.... 2 ..... I

c

'

"r ' 'i~

h.

ep

=,,,J,]JrJ,l,,,J=,

12!

lJlllJt

Li

[ °

~

Fig. 3. Responses to stimulation of the carotid sinus nerve (CSN) in a decerebrate cat. Stimulus trains (25 Hz, 0.5 ms duration, 30 s train) are indicated by horizontal bars. During CSN stimulation, integrated hypoglossal (XII) and phrenic (Phr) activities and arterial blood pressure (BP) were increased; the increase in blood pressure was uncharacteristically large in this cat. Following stimulation, phrenic activity returned rapidly to its prestimulus control value. The amplitude of respiratory discharge in hypoglossal motoneurons remained elevated following stimulation, and only slowly returned to prestimulus values. There were no significant effects on burst frequency, paCO z did not change significantly following CSN stimulation.

U

lOnA

lJJlJ=J=JJti

[

100mmHg

/l

. l min

-,0 x.

I'

u

i!i!!i! ~,

,

Phr

_/L

~

Ir /~. /~L /'~.

L~_ 0my

/~__/L_

2ms

Fig. 4. Examples of intrac¢llular recordings from hypoglossal motoneurons with central respiratory drive potentials (CRDPs). In A, SLN stimulation (indicated by horizontal bars) caused strong depolarization as well as poststimulus augmentation of tonic membrane potential and CRDPs (reflecting patterns in the integrated hypogiossal nerve). In B, recording from another neuron is shown with an expanded time scale; neither depolarizing (+15 nA) nor hyperpolarizing (-15 nA) intracellular current pulses had any effect on resting membrane potential or the amplitude of CRDPs. In G, two superimposed antidromic action potentials recorded from the neuron shown in B following stimulation of the hypoglossal nerve. MP, membrane potential; GM, current monitor. Other abbreviations as in Fig. 1.

220 reset the magnitude of C R D P s following SLN stimulation to prestimulus levels via intracellular hyperpolarizing current pulses in any neuron. The general activity pattern of these neurons resembled the behavior of the whole hypoglossal nerve (Fig. 4A). W h e n direct depolarizing or hyperpolarizing pulses were injected into these neurons, plateau potentials or other prolonged effects could not be detected (Fig. 4B), regardless of the size of the depolarizing pulse, the bias current or the prevailing level of paCO2 • O t h e r neurons (n = 27) were either unaffected by SLN stimulation, o r were activated, but without effects lasting b e y o n d the stimulus train (Table I). These neurons were most often without respiratory modulation. F u r t h e r m o r e , there were no consistent long lasting effects following either direct depolarizing or hyperpolarizing current pulses, regardless of the bias current. Nevertheless, the hypoglossal nerve showed prolonged augmentation of

respiratory discharge following SLN stimulation during these observations. Thirteen neurons were not tested regarding their response to SLN stimulation before the recording was lost, but they were tested with direct current pulses. Of all m o t o n e u r o n s tested with intracellular depolarization, only 4 trials a p p e a r e d to trigger p r o l o n g e d effects on m e m b r a n e potential (two not responsive to SLN stimulation, two untested); however, in each case, hyperpolarizing current did not reverse the effect, suggesting that the a p p a r e n t effect was due to recording instability.

Morphological identification of serotonergic input Serotonin immunoreactive boutons were found throughout the hypoglossal nucleus with a higher density in the caudal and dorsal part. A n example of a transverse section through the hypoglossal nucleus following serotonin immunohistochemistry is shown in Fig. 5A; a reconstruction of an

C

Fig. 5. Close appositions of serotonin immunoreactive boutons with an HRP labeled hypoglossal motoneuron. A: transverse section of the hypoglossal nucleus (approx. 1.00 mm caudal to the obex) showing a moderately dense network of serotonin immunoreactive fibers. Such fibers were found throughout the nucleus, with a higher density in the caudal and dorsal regions. B: a microphotograph showing an example of a close apposition (open triangle) of serotonin immunoreactive bouton with an HRP labeled dendrite of the hypoglossal motoneuron. Close appositions were sparse and were more prevalent on distal dendrites. The photographed area is shown as an obliquely positioned box in C. C: 3-dimensional reconstruction of the hypoglossai motoneuron in the sagittai plane. The dotted line indicates the dorsal border of the hypoglossal nucleus. The neuron exhibited CRDPs, was strongly activated by SLN stimulation and showed augmentation during the poststimulus period. D: camera lucida drawing of serotonin immunoreactive boutons, some of which form close appositions with the dendrites of the labeled neuron. The reconstructed area is shown in C (horizontally oriented box).

221 HRP-filled hypoglossal motoneuron that exhibited CRDPs and prolonged augmentation of the CRDPs following SLN stimulation is illustrated in Fig. 5C. Eight hypoglossal motoneurons were filled intracellularly with HRP (cf. ref. 34); 5 of these revealed augmentation of the CRDPs following SLN stimulation. Some close appositions of serotonergic boutons with HRP-filled cells were found, as shown in Fig. 5B,D, and these appositions appeared to be most frequent near the distal dendrites. However, a comparison with another study performed in this laboratory using similar criteria to examine serotonergic input to phrenic motoneurons2s shows that the number of appositions between serotonin immunoreactive boutons and HRP filled hypoglossal motoneurons is relatively sparse. This suggests that serotonin plays a less significant role in modulating the activity of hypoglossal motoneurons with CRDPs. DISCUSSION The major finding of this study is that both superior laryngeal and carotid sinus nerve stimulations elicit a form of memory in the respiratory discharge of hypoglossal motoneurons, lasting up to 10 min. Although the mechanism of this prolonged augmentation in hypoglossal discharge is unclear, it appears to result from an increased CRDP in hypoglossal motoneurons with only occasional elevations in tonic membrane potential (3/18). We were not able to elicit convincing plateau potentials with direct depolarizing currents in any cell and it does not appear that serotonin is necessary for expression of prolonged augmentation of respiratory discharge in hypoglossal motoneurons. Thus, the mechanism of prolonged augmentation does not appear similar to long term facilitation of phrenic motoneurons following carotid sinus nerve stimulation 26 nor long lasting plateau potentials in extensor lumbar motoneurons 6, both of which require serotonin. It appears more likely that prolonged augmentation of respiratory discharge in hypoglossal motoneurons results from a mechanism similar to short term potentiation 42 or afterdischarge in phrenic motoneurons following CSN stimulation (cf. ref. 10).

Critique of methods Decerebrate and decerebellate, rather than anesthetized cats were used in this study for two reasons. First, hypoglossal motoneurons are depressed by anesthesia to a greater relative extent than spinal respiratory motoneurons (cf. ref. 18). Thus, respiratory related discharge in hypoglossal motoneurons occurs at lower levels of chemoreceptor drive in decerebrate versus anesthetized cats 16. Second, serotonergic brainstem neurons are more active in decerebrate cats, allowing manifestation of an

intrinsic membrane property expressed as long lasting plateau potentials (ref. 6; see below for additional discussion). Such plateau potentials do not occur in the motoneurons of cats anesthetized with barbiturates or ketamine. Thus, it was essential to use decerebrate cats since one of our initial objectives was to determine if long lasting plateau potentials occur in hypoglossal motoneurons. One drawback of decerebrate preparations is that phrenic motoneuron activity saturates at relatively low levels of chemoreceptor drive (ref. 9; unpublished observations). A number of feedback loops were left intact in our preparation including feedback from vagal stretch receptors, contralateral carotid chemoreceptors and baroreceptors, as well as central chemoreceptors. Effects from vagal stretch receptors, such as entrainment of respiratory rhythm were minimized by ventilating the cats with a high frequency and low tidal volume. Obvious forms of entrainment with the pump were not apparent in any animal. However, since vagal efferent fibers were intact, possible effects of SLN or CSN stimulation on airway smooth muscle could stimulate slowly adapting pulmonary stretch receptors 11 thereby affecting our results. Nevertheless, stretch receptor activation from smooth muscle constriction is not expected to result in prolonged activation of hypoglossal nerve discharge since pulmonary stretch receptors strongly inhibit hypoglossal motoneurons (cf. ref. 18). It remains possible that indirect stimulation of pulmonary stretch receptors accounts for the slowing of phrenic bursts following SLN stimulation. Both CSN and SLN stimulation increased blood pressure to varied degrees, potentially affecting ventilatory activity via baroreceptors or changes in pulmonary gas exchange (acting indirectly via chemoreceptors). However, increased baroreceptor activity inhibits rather than excites hypoglossal nerve activity (cf. ref. 31). To account for possible changes in paCO2 or paO2 due to changes in pulmonary gas exchange, we were careful to monitor blood gas levels before and after trains of stimuli. If blood gas composition changed, trials of nerve stimulation were not considered further. This is an essential issue since chemoreceptor activation has a disproportionately large influence on hypoglossal relative to phrenic motoneurons (cf. refs. 18, 31). Electrical stimulation of CSN and SLN cannot be considered the equivalent of physiological stimulation of peripheral chemoreceptors or upper airway receptors. Almost all fibers are activated simultaneously with a high frequency relative to physiological levels of discharge. It is reassuring that the reflex responses closely resemble the expected effects of carotid chemoreceptor and upper airway receptor activation resulting from mechanical or chemical stimuli (cf. refs. 18, 31). It should be noted that

222 upper airway receptors also elicit swallowing and other tongue reflexes not necessarily related to respiration (cf. ref. 23). Two techniques were used to investigate the role of endogenous serotonin in modulating the activity of hypoglossal motoneurons with CRDPs. First, combined intracellular labeling and immunohistochemistry were used to reveal the presence of 5-HT containing terminals in the vicinity of the motoneurons. This technique was used to qualitatively assess the presence of 5-HT terminals near motoneurons, and does not allow strong statements about their functional significance. For example, strategic positioning of 5-HT boutons along the somato-dendritic tree may impart functional significance beyond their number or density. Second, methysergide was used to antagonize endogenous serotonin receptors. Methysergide antagonizes a broad range of 5-HT receptor subtypes, but we have no direct test that methysergide was effective under our experimental conditions. However, similar doses to those used in this study are effective in blocking both long term facilitation 26 and plateau potentials 6"15 in cats. Furthermore, cardiopulmonary depression following administration of methysergide indicates that the drug was effective, an observation consistent with previous reports 26.

stimulation in adult cats as previously reported by Gerber and Polosa ~2. This inhibition was restricted to a decrease in phrenic burst frequency without consistent effect on burst amplitude. Furthermore, the absolute change in burst frequency following stimulation is a function of the prestimulus frequency in each cat, a previously unreported finding. In contrast with the inhibitory effects of SLN stimulation on spinal respiratory motoneurons, motoneurons innervating upper airway muscles are strongly activated during stimulation (cf. refs. 3, 8, 31). There are a few recent accounts suggesting that the effects of SLN stimulation persist for brief periods in both hypoglossal m o t o n e u r o n s 22"23"41 and facial motoneurons 19. The present study demonstrates prolonged effects on hypoglossal respiratory discharge of longer duration and greater magnitude with more intense SLN stimulation. Although quite different in their effects on phrenic nerve activity, CSN and SLN stimulation have similar effects on hypoglossal discharge in both magnitude and time course. Thus, although SLN and CSN stimulation may share a common mechanism of action on hypoglossal motoneurons (see below), prolonged augmentation of respiratory discharge in hypoglossal motoneurons must occur in different neuronal pathways than afterdischarge in phrenic motoneurons following CSN stimulation.

Long lasting effects of SLN and CSN stimulation The most thoroughly documented example of respiratory memory concerns the facilitatory actions of CSN activation on the phrenic neurogram (cf. ref. 10). Results of the present study did not reveal afterdischarge or long term facilitation in the phrenic neurogram following CSN stimulation. This finding is not completely in disagreement with previous reports since conditions were established that result in respiratory modulation of the hypoglossai nerve, and were relatively hypercapnic with respect to the apneic threshold of phrenic activity. Both afterdischarge 7 and long term facilitation 25 are most evident when paCO2 is only slightly above the threshold for rhythmic phrenic activity. The magnitude of afterdischarge is greatly diminished in hypercapnia 7 or hypocapnia 8, although it appears that the fundamental mechanism underlying this phenomenon is still operative (cf. ref. 10). Thus, prolonged augmentation of phrenic discharge following CSN stimulation may have been obscured in the present study due to relative hypercapnia. In contrast, SLN activation inhibits the phrenic neurogram and this inhibition outlasts the period of stimulation (cf. refs. 10, 18). This prolonged effect is most pronounced in infants and becomes less prominent as the animal matures 2°'21'38. Our results confirm that prolonged inhibition of phrenic discharge follows SLN

Mechanisms Withington-Wray et al. 4~ demonstrated that SLN and CSN stimulation both elicit complex patterns of excitatory and inhibitory postsynaptic potentials in respiratory modulated hypoglossal motoneurons. Excitatory potentials predominate during trains of stimulation 24. The focus of the present study concerned SLN stimulation and direct depolarizing current pulses on resting membrane potential and the magnitude of CRDPs. Huitborn and colleagues 5'6'15 recently reported an intrinsic membrane property of lumbar motoneurons termed bistable state behavior, leading to plateau potentials and prolonged excitability following either synaptic or direct depolarization. This property requires the presence of monoamines (serotonin in extensor motoneurons, norepinephrine in flexors) and can be reversed by synaptic inhibition or direct hyperpolarization. The results of this study provide little evidence for the existence of bistable state behavior in hypoglossal motoneurons, nor that it underlies the mechanism of prolonged augmentation in hypoglossal motoneuron discharge following SLN or CSN stimulation. First, stimulation of SLN did not elicit prolonged, tonic depolarization of the membrane potential in most cells (15/18). In contrast, CRDPs were increased, most commonly at the same resting membrane potential. Second,

223 synaptic inhibition of hypoglossal nerve activity by stimulation of the vagus or intercostal nerves was ineffective in resetting augmented hypoglossal nerve discharge due to SLN stimulation, although it is not clear that these inputs actively inhibit hypoglossal motoneurons (i.e. elicit inhibitory postsynaptic potentials). Finally, direct depolarizing or hyperpolarizing current pulses did not elicit transitions in the excitability of hypoglossal motoneurons as they do in lumbar motoneurons. Nevertheless, it remains possible that intracellular current pulses altered the membrane potential near the soma only and did not project along the dendrites. Depolarization in distal dendrites may be necessary to elicit changes in hypoglossal motoneuron excitability. Since serotonin is necessary for long term facilitation of phrenic discharge following CSN stimulation26 and for bistable state behavior in extensor lumbar motoneurons 15, we investigated the role of serotonin in prolonged augmentation of hypoglossal discharge following SLN stimulation. Although evidence was found that serotonergic boutons form close appositions to characterized hypoglossal motoneurons, their density appeared very low in comparison to putative serotonin contacts with motoneurons of the phrenic nucleus28 or nucleus ambiguus TM. It is hazardous to ascribe function based on qualitative estimates of serotonergic input (see above); nevertheless, our inability to demonstrate plateau potentials or the equivalent of long term facilitation in hypoglossal motoneurons is consistent with the hypothesis that direct 5-HT effects are minimal in this motoneuron pool. Furthermore, the inability to block prolonged augmentation of hypoglossal discharge following SLN stimulation with methysergide suggests that serotonin does not play a mandatory role in the mechanism underlying this response. The magnitude, time course, paCO2 dependence, common response to several afferent inputs and lack of methysergide sensitivity suggest that the mechanism of prolonged augmentation in hypoglossal discharge has a similar mechanism to afterdischarge in phrenic motoneurons (cf. ref. 10), although in different neural pathways. The mechanism of afterdischarge is unknown, but it may result from presynaptic Ca 2+ uptake similar to short term potentiations42 or a network of brainstem neurons that retains a degree of enhanced activity once stimulated and provides increased synaptic input to medullary premotoneurons (cf. ref. 10). The specific neural pathways acting on hypoglossal motoneurons must differ from those acting on bulbospinal neurons in the dorsal and ventral respiratory groups since SLN stimulation has opposite effects on phrenic vs hypoglossal nerve discharge. Anatomical studies suggest that hypoglossal motoneurons receive extensive inputs from the adjacent reticular

formation2"39. Furthermore, neurons of the reticular formation in the vicinity of the hypoglossal nucleus receive inputs from both glossopharyngeal and superior laryngeal nerves 33'37. St. John 36 demonstrated that stimulation of reticular formation can elicit greater effects on upper airway than on spinal motoneurons, and sometimes elicits prolonged augmentation of respiratory discharge in hypoglossal motoneurons lasting several respiratory cycles. Thus, SLN stimulation may activate neurons in the reticular formation near the hypoglossal nucleus, eliciting mechanisms causing prolonged activation of hypoglossal motoneurons with respiratory modulation via increased synaptic input. It is doubtful that these same reticular neurons have bulbospinal projections since phrenic motoneurons are either unaffected or are actually inhibited during this time.

Physiological significance Sensory receptors in the SLN and CSN are likely to play an important role in maintaining upper airway patency during conditions such as sleep or anesthesia when upper airway tone is diminished (cf. refs. 18, 31). Respiratory modulated contractions in upper airway muscles are necessary to offset negative pressures exerted on the flaccid upper airways by thoracic respiratory muscles (cf. refs. 18, 31). Furthermore, once airway collapse occurs, surface adhesive forces complicate the task of restoring patency to the airways. The genioglossus muscle of the tongue is particularly susceptible to collapse (cf. ref. 40). During upper airway occlusion, negative (and positive) pressures stimulate laryngeal receptors which, through the reflex action, augment hypoglossal nerve discharge in an effort to restore airway patency 17'22'23'32. The inhibitory actions of SLN receptor stimulation on phrenic nerve activity are complimentary since they temporarily halt negative pressures generated by breathing movements. Once airway patency is restored, it is important to prevent any recurrence. Therefore, the combination of persistent augmentation in respiratory hypoglossal activity, as revealed in the present study, and persistent inhibition of respiratory frequency may play a protective role in maintaining airway patency for prolonged periods in physiological circumstances that predispose to airway collapse. Prolonged augmentation of the integrated genioglossus EMG can be seen following more physiological activation of upper airway receptors 23. If occlusive apnea persists despite activation of laryngeal receptors, slower changes in arterial blood gases will stimulate chemoreceptors, activating respiratory discharge in hypoglossal motoneurons further. Under these conditions, where life is threatened, both inspiratory and expiratory thoracic respiratory motoneurons would be

224 activated 35 in an effort to force the airway open and restore ventilation. Once again, persistent augmentation of hypoglossal discharge would be of advantage in maintaining airway patency once the airway had been reopened and receptor inputs had returned to normal. Although interactions between superior laryngeal stimulation and chemoreceptor drive have been reported for overall ventilation27, similar studies have not been done concerning interactions between SLN and CSN afferent inputs in modulating upper airway muscles. Such studies would be of considerable interest with regard to both the period of stimulation and the poststimulus period. It is also possible that afferent fibers mediating prolonged effects of SLN stimulation on hypoglossal and phrenic nerve activities are chemoreceptive, sensing water, milk or other foreign substances in the upper airways (cf. ref. 31). Once upper airway chemoreceptors are activated, inhibiting phrenic activity and augmenting REFERENCES 1 Aides, L.D., Chronister, R.C., Marco, L.A., Haycock, J.W. and Thibault, J., Differential distribution of biogenic amines in the hypoglossal nucleus of the rat, Exp. Brain Res., 73 (1988) 305-314. 2 Amri, M. and Car, A., Projections from the medullary swallowing center to the hypoglossal motor nucleus: a neuroanatomical and electrophysioiogical study in sheep, Brain Research, 441 (1988) 119-126. 3 Barlett, Jr., D., Respiratory functions of the larynx, Physiol. Rev., 69 (1989) 33-178. 4 Brouillette, R.T. and Thach, B.T., Control of genioglossus muscle inspiratory activity, J. Appl. Physiol., 49 (1980) 801-808. 5 Conway, B.A., Hultborn, H., Kiehn, O. and Mintz, I., Plateau potentials in alpha motoneurons induced by intravenous injection of L-DOPA and clonidine in the spinal cat, J. Physiol., 405 (1988) 369-384. 6 Crone, C., Hultborn, H., Kiehn, O., Mazieres, L. and Wigstr6m, H., Maintained changes in motoneuronal excitability by short-lasting synaptic inputs in the decerebrate cat, J. Physiol., 405 (1988) 321-343. 7 Eldridge, EL., Central neural respiratory stimulatory effect of active respiration, J. Appl. Physiol., 37 (1974) 723-735. 8 Eldridge, EL., Subthreshold central neural respiratory activity and afterdischarge, Resp. Physiol., 39 (1980) 327-343. 9 Eldridge, EL., Gill-Kumar, P. and Millhorn, D.E., Inputoutput relationships of central neural circuits involved in respiration in cats, J. Physiol., 311 (1981) 81-95. 10 Eldridge, EL. and Millhorn, D.E., Oscillation, gating, and memory in the respiratory control system. In A.P. Fishman (Ed.), Handbook of Physiology, Section 3, Vol. 2, American Physiol. Soc., Bethesda, MD, 1986, pp. 93-114. 11 Fisher, J.L., Sant'Ambrogio, F.B. and Sant'Ambrogio, G., Stimulation of tracheal slowly adapting stretch receptors by hypercapnia and hypoxia, Resp. Physiol., 53 (1983) 325-339. 12 Gerber, U. and Polosa, C., Some effects of superior laryngeal nerve stimulation on sympathetic preganglionic neuron firing, Can. J. Physiol., 57 (1979) 1073-1081. 13 Hanker, J.R., Yates, P.E., Metz, C.B. and Rustioni, A., A new specific, sensitive and non-carcinogenic reagent for the demonstration of horseradish peroxidase, Histochem. J., 9 (1977) 789-792. 14 Holtman, Jr., J.R., Immunohistochemical localization of serotonin- and substance P-containing fibres around respiratory muscle motoneurons in the nucleus ambiguus of the cat,

hypoglossal nerve discharge, prolonged inhibition of phrenic activity may be an adaptive response since diminished respiratory airflow is less likely to cause aspiration of the foreign substance. O n the other hand, prolonged inhibition of ventilatory activity following chemoreceptor stimulation may be disadvantageous, leading to severe hypoventilation at a time when the respiratory system is already compromised. Perhaps such effects help to explain severe ventilatory depression and even mortality observed in young monkeys when milk contacts the upper airways 3s.

Acknowledgements. This work was supported by a grant from the AMRE GSM was supported by a Research Career Development Award from the National Institutes of Health (HL 1494) and CJ by the Wellcome Trust (U.K.). We would like to thank V. Yardley and K. Bark for excellent technical assistance, and Dr. P. Pilowsky for advice concerning immunohistochemistry. We would also like to thank Sandoz Pharma Ltd for their generous gift of methysergide. Neuroscience, 26 (1988) 169-178. 15 Hounsgaard, J., Hultborn, H., Jespersen, B. and Kiehn, O., Bistability of alpha motoneurons in the decerebrate cat and in the acute spinal cat after intravenous 5-hydroxytryptophan, J. Physiol., 405 (1988) 345-367. 16 Hwang, J.-C., St. John, W.M. and Bartlett, Jr., D., Respiratoryrelated hypoglossal nerve activity: influence of anesthetics, J. Appl. Physiol., 55 (1983) 785-792. 17 Hwang, J-C., St. John, W.M. and Bartlett, Jr., D., Afferent pathways for hypoglossal and phrenic responses to changes in upper airway pressure, Resp. Physiol., 55 (1984) 341-354. 18 Iscoe, S.D., Central control of the upper airway. In O.P. Mathew and G. Sant'Ambrogio (Eds.), Respiratory Function of the Upper Airway, Dekker, New York, 1988, pp. 125-192. 19 Kukwa, A. and Janczewski, W.A., Effects of superior laryngeal nerve (SLN) stimulation on the activity of the facial nerve, Proc. XXXI International Congress of Physiological Sciences, 1989, p. 1396. 20 Lawson, E.E., Prolonged central respiratory inhibition following reflex-induced apnea, J. Appl. Physiol., 50 (1981) 874-879. 21 Lawson, E.E., Recovery from central apnea: effect of stimulus duration and end-tidal CO 2 partial pressure, J. Appl. Physiol., 53 (1982) 105-109. 22 Mathew, O.P., Abu-Osba, U.K. and Thach, B.T., Influence of upper airway pressure changes on genioglossal muscle respiratory activity, J. Appl. Physiol., 52 (1982) 438-444. 23 Mathew, O.P., Abu-Osba, Y.K. and Thach, B.T., Genioglossus muscle responses to upper airway pressure changes: afferent pathways, J. Appl. Physiol., 52 (1982) 445-450. 24 Mifflin, S.W., Frequency dependence of superior laryngeal nerve (SLN) input to inspiratory hypoglossal motoneurons (IHMs), FASEB J., 3 (1989) 149. 25 Millhorn, D.E., Eldridge, EL. and Waldrop, T.G., Prolonged stimulation of respiration by a new central neural mechanism, Resp. Physiol., 41 (1980) 87-103. 26 Millhorn, D.E., Eldridge, F.L. and Waldrop, T.G., Prolonged stimulation of respiration by endogenous central serotonin, Resp. Physiol., 42 (1980) 171-188. 27 Nishino, T., Effects of chemical drive on the inhibition of respiration produced by laryngeal stimulation in cats, Jap. J. Physiol., 30 (1980) 827-840. 28 Pilowsky, P.M., de Castro, D., Llewellyn-Smith, I., Lipski, J. and Voss, M., Serotonin immunoreactive boutons make synapses with feline phrenic motoneurons, J. Neurosci., 10 (1990) 1091-1098.

225 29 Reed, W.R., Roberts, J.L. and Thach, B.T., Factors influencing regional patency and configuration of the human infant upper airway, J. Appl. Physiol., 58 (1985) 635-644. 30 Remmers, J.E., deGroot, W.E., Sauerland, E.K. and Anch, A.M., Pathogenesis of upper airway occlusion during sleep, J. Appl. Physiol., 44 (1978) 931-938. 31 Sant'Ambrogio, G. and Mathew, O.P., Control of upper airway muscle, NIPS, 3 (1988) 167-172. 32 Sant'Ambrogio, G., Mathew, O.P., Fisher, J.T. and Sant'Ambrogio, F.B., Laryngeal receptors responding to transmural pressure, airflow and local muscle activity, Resp, Physiol., 54 (1983) 317-330. 33 Sessle, B.J., Excitatory and inhibitory inputs to single neurons in solitary tract nucleus and adjacent reticular formation, Brain Research, 53 (1973) 319-331. 34 Shohara, E. and Dostrovsky, J.O., Morphology of normal cat hypoglossal motoneurons as determined by intracellular HRP injection, Soc. Neurosci. Abstr., 10 (1984) 748. 35 Smith, C.A., Ainsworth, D.M., Henderson, K.S. and Dempsey, J.A., Differential responses of expiratory muscles to chemical stimuli in awake dogs, J. Appl. Physiol., 66 (1989) 384-391.

36 St. John, W.M., Influence of reticular mechanisms upon hypoglossal trigeminal and phrenic activities, Resp. Physiol., 66 (1986) 27-40. 37 Sumino, R. and Nakamura, Y., Synaptic potentials of hypoglossai motoneurons and a common inhibitory interneuron in the trigemino-hypoglossal reflex, Brain Research, 73 (1974) 439454. 38 Sutton, D., Taylor, E.M. and Lindemann, R.C., Prolonged apnea in infant monkeys resulting from stimulation of superior laryngeal nerve, Pediatrics, 61 (1978) 519-527. 39 Takada, M., Itoh, K., Yasui, Y., Mitani, A., Nomura, S. and Mizuno, N., Distribution of premotor neurons for the hypoglossal nucleus in the cat, Neurosci. Len., 52 (1984) 141-146. 40 Van Lunteren, E. and Strohl, P., The muscles of the upper airways, Clin. Chest Med., 7 (1986) 171-188. 41 Withington-Wray, D.J., Mifflin, S.W. and Spyer, K.M., Intracellular analysis of respiratory-modulated hypoglossal motoneurons in the cat, Neuroscience, 25 (1988) 1041-1051. 42 Zucker, R.S., Short term synaptic plasticity, Annu. Rev. Neurosci., 12 (1989) 13-31.