- Email: [email protected]
The effects of hypercapnia and hypoxia on single hypoglossal nerve fiber activity
Respiration Physiology, (1983) 54, 55-66
55
Elsevier
T H E EFFECTS OF H Y P E R C A P N I A A N D H Y P O X I A O N S I N G L E H Y P O G L O S S A L N E R V E FIBER ACTIVITY
J Y O T I M I T R A and N E l L S. C H E R N I A C K Department of Medicine, Case Western Reserve University, Cleveland, OH 44106, U.S.A.
Abstract. The respiratory related modulation of hypoglossal nerve activity has been studied at the single fiber level in cats under hyperoxic hypercapnia and hypoxic conditions and their conduction velocities determined. Changes in fiber activity were compared to simultaneous changes occurring in phrenic activity. Three different kinds of discharge patterns were observed: (a) inspiratory, (b) phasic activity during both inspiration and expiration, and (c) continuous random activity with no respiratory modulation. These fibers could be grouped into three categories according to their pattern of discharge during CO 2 breathing. Type I fibers, mean conduction velocity of 30.0 m/sec, exhibited only an inspiratory phasic discharge during 100~o 02 breathing. Their discharge frequency increased rapidly with higher levels of CO 2 and hypoxia. Type II fibers, mean conduction velocity of 36.7 m/sec, had three different kinds of inspiratoryexpiratory discharge patterns during 100~ 02 breathing. With increasing hypercapnia or hypoxia fibers of this group discharged phasically during inspiration and discharged at low frequency during expiration. Type III fibers had a non phasic discharge pattern at 100~o 02 breathing and at all levels of CO 2 tested (up to 10~). Discharge frequency rose during CO 2 rebreathing and hypoxia, but the rate of increase was much less than Type I and Type II fibers. Their mean conduction velocity was 41.3 m/sec. The inspiratory activity of Type I and II fibers increased their activity more than the phrenic during hypercapnia and hypoxia. Type I1 and Type III fibers are responsible at least in part for the tonic activity of the nerve. Cat Control of breathing Hypercapnia Hyperoxia
Hypoglossal nerve Hypoxia Nerve fiber Phrenic nerve
Accepted Jbr publication 4 July 1983 Address correspondence to: Dr. Jyoti Mitra, Department of Medicine, University Hospitals, 2074 Abington Road, Cleveland, OH 44106, U.S.A. 0034-5687/83/$03.00 © 1983 Elsevier Science Publishers B.V.
56
J. MITRA AND N.S. CHERNIACK
The activity of many of the cranial nerves that supply the upper airway muscles is modulated by respiration. This activity appears to be especially important during sleep in maintaining upper airway patency (Brouillette and Thach, 1980). Loss of tonic and phasic activity in laryngeal muscles and oral muscles such as the genioglossus is believed to lead to obstruction during inspiration in some sleeping humans (Onal et al., 1981). The changes in genioglossus muscle activity that occur with respiration have been investigated in both animals and humans (Andrew, 1955; Blom, 1960; Brouillette and Thach, 1980; Lei, 1961; Lowe and Sessel, 1973; Mitchinson and Yoffey, 1947; Miller and Bowman, 1974; Onal et al., 1981). Studies from our laboratory (Weiner et al., 1980, 1982) have indicated that the integrated hypoglossal nerve activity (medial branch) - - the motor nerve to genioglossus muscle - - has both phasic and tonic activity during respiratory drive. While the tonic activity is linearly related to phrenic nerve activity, the phasic activity tends to increase their activity more than that of the phrenic nerve during hypercapnia and hypoxia. The purpose of our present investigation was two-fold : (1) to study whether the phasic and tonic activity of the integrated whole nerve could be related to discharge patterns occurring in a single fiber preparation; and (2) to determine how hypercapnia and hypoxia affects different single fibers.
Materials and methods
Acute experiments were performed on 15 cats of both sexes (1.54.5 kg body weight) which were anesthetized with alpha chloralose (36 mg/kg) administered intraperitoneally. Local anesthesia induced by 2 ~ Lidocaine (Xylocaine) was used over pressure points and in incised tissue. Body temperature was maintained at 38 _+ 1 °C using an electric thermal blanket. The saphenous vein of one forelimb was cannulated for infusion of saline and Flaxedil (4 mg/kg). The trachea was intubated for artificial ventilation and to allow the insertion of a capnograph (GodartStatham) probe to monitor end-tidal CO2. Through a midline incision, extending from the symphysis of the lower jaw to the manubrium, both the hypoglossal (medial branch) and the fifth root of the phrenic nerve were exposed ipsilaterally. They were then separated from the surrounding tissues and severed at their most peripheral point. The former was used for a single nerve fiber preparation and the latter for monitoring central respiratory activity. Both vagus nerves were also isolated and cut in order to eliminate stretch receptor influences on hypoglossal motoneurons. The cutaneous edges of the incision were then elevated to make a trough which was filled with warmed mineral oil. The oil temperature was maintained at body temperature with the aid of a small warm water circulating coil immersed in the pool. The conduction velocity of a single fiber was measured according to the method described by Paintal (1953) with slight modification. In short, the connective tissue
S I N G L E H Y P O G L O S S A L NERVE FIBER ACTIVITY
57
sheath of the hypoglossal nerve was split and desheathed to a length of 6 cm. The most central part of the desheathed nerve was placed on a pair of stainlesssteel stimulating electrodes for stimulation later. The peripheral end of the nerve was then divided longitudinally into several smaller strands using an iridectomy knife. Under microscopic control, using a pair of sharpened dissecting needles, one' of the smaller strands was further dissected and placed on a monopolar stainless-steel recording electrode of 200 #m in diameter. An indifferent electrode was placed in the neck muscle. The distance between the stimulating and the recording electrodes was 6 cm. The activity of the strand was amplified (Grass Instruments, AC Preamplifier, P51 l) and fed in parallel to one channel of a dualbeam oscilloscope (Tektronix, 5113), an audio amplifier and to a tape recorder (Hewlett-Packard, 3968A). The nerve was then stimulated with rectangular pulses delivered by a stimulator (Grass Instruments, $48) connceted to the stimulating electrodes via an isolation unit (Grass Instruments, PSIU 6). The action potential so recorded was compound in nature. The strand was subdivided further until a single action potential could be obtained by stimulation. The conduction distance of the action potential in the selected strand was measured with a pair of dividers from the cathode of the stimulating electrodes to the recording electrode. Spontaneous discharge of the fibers thus isolated were also used to study the effects of hypercapnia and hypoxia. The fifth cervical root of the phrenic nerve (ipsilateral to the hypoglossal nerve) was cut peripherally and desheathed. The desheated nerve was then placed on a pair of stainless-steel hook electrodes (200 #m in diameter), immersed in warm mineral oil at the animal's body temperature. The output of the electrode was amplified (Grass Instuments, AC Preamplifier, P511), rectified and processed by a Paynter filter (time constant 200 msec) to obtain a moving average signal of the phrenic nerve and thereby to assess central respiratory drive. The moving average signal was connected to the second channel of the dual-beam oscilloscope to monitor the temporal relationship of hypoglossal unit discharge to the phrenic signal. The moving average signal was also stored on the magnetic tape. Recordings were made after the animals breathed 100~o 02 for 10 min, and then under conditions of increased chemical drive, progressive hyperoxic hypercapnia, and isocapnic hypoxia. Progressive hyperoxic hypercapnia was produced in the following way. A mixture of 5~o CO2 and 95~ 02 was delivered to the inspiratory port of the ventilator via a 2 L Collins bag. Gas from the expiratory line was then returned to the bag and the resulting mixture was rebreathed. The end-tidal CO2 was continuously measured with an infrared CO2 analyzer (Godart-Statham), and recorded on magnetic tape. Hypoxia was produced by connecting the inlet port of the ventilator to a 5 L Collins bag containing 12~ 02 and 88~ N2. The expiratory port of the ventilator remained open to atmosphere. Data were analyzed from the magnetic tape in the following way. First, tape segments were identified with different CO2 levels. Then the integrated phrenic
58
J. MITRA AND N. S. CHERNIACK
neurogram was displayed in one channel of a signal averager (Princeton Applied Research, 4202) and the averager was triggered by the inspiratory cycle of the phrenic nerve at normal averaging mode. Hypoglossal single fiber activity was processed by a Schmitt trigger circuit in order to convert fiber activity into uniform pulses of fixed amplitude and duration. The output pulses of the Schmitt trigger were then fed into the second channel of the averager. In this way the temporal relationship between the two signals were obtained at different endtidal CO2 levels. Counting of discharge frequency during inspiratory phase was made possible with the use of a comparator circuit and a gate-operated digital pulse counter (Charles Ward Enterprises). The comparator was set in such a way that during inspiration when voltage of the integrated phrenic neurogram rose above the baseline activity the comparator was turned on and remained so during the rest of the inspiratory phase. At the onset of expiration the comparator was turned off. The output signal from the comparator was used to open (during inspiration) and close (during expiration) the gate of the counter. In this way the counter counted spike activity only during the inspiratory phase of the respiratory cycle. Mean discharge rate during inspiratory phase was calculated dividing the total number of spikes during inspiration by the inspiratory time (TI). For observation of single fiber activity pattern, it was displayed on the oscilloscope by intensifying beam with 'z' axis modulation. Statistical analyses (analysis of variance) were also done to test the significance of discharge rate among the three types of fibers.
Results
Fiber responses in cats breathing oxygen A total of forty-two fibers were used in the study. In general during 100% 02 breating (end-tidal (ET) CO2 4.0~o) relatively few fibers were found phasically active. However, a close inspection of discharge patterns with respect to integrated phrenic nerve activity at 100~o 02 breathing revealed a variety of discharge patterns. Some of the discharge patterns we observed were the following. (a) Inspiratory. Fiber activity was restricted only to the inspiratory phase of the respiratory cycle. Such fibers were silent during the expiratory phase (fig. 1A). (b) Inspiratory-Pause-Expiratory (Burst). Frequency of discharge increased during inspiratory phase. However, at the peak of inspiration there was a pause. As expiration started there was a short burst of activity. Such fibers did not have activity during the rest of the expiratory phase (fig. 1B). (c) Inspiratory-Pause-Expiratory. These fibers did not have burst activity during expiration. The activity increased during inspiration and at the peak of inspiration there was a pause followed by resumption of continuous activity at a lower rate during the expiratory phase (fig. 1C).
59
SINGLE HYPOGLOSSAL NERVE FIBER ACTIVITY
A
B
C
1.0sec.
D
E
Fig. 1. Different discharge patterns observed during oxygen breathing. The upper trace of each frame shows the raw hypoglossal single fiber activity displayed on an oscilloscope by 'Z' axis modulation of
its beam intensity. The lower tracing shows the integrated phrenic nerve activity. All tracings are single sweep. A. Activity only during inspiration. B. Inspiratory activity as in A, followed by a pause and then a short burst of activity during expiration. C. Inspiratory activity followed by a pause as in B, but activity was resumed at a lower rate during expiration. There was no burst activity during expiration. D. Higher activity during inspiration, followed by lower activity during expiration without any pause. E. Random activity not changing with inspiration or expiration.
(d) Continuous-Peak frequency in &spiration. These fibers had continuous activity through the respiratory cycle and had no pause at the peak of inspiration (compare with fig. 1C). However, the activity increased during inspiration and decreased during expiration (fig. 1D). (e) Continuous, no respiratory modulation. These fibers did not have respiratory modulation and the discharge patterns were continuous and random (fig. 1E).
Effects ofhypercapnia and hypoxia.
The single fibers could be divided into three types based on their responses to hypercapnia and hypoxia as shown in fig. 2. Figure 2 displays processed hypoglossal single fiber activity together with the integrated phrenic nerve activity averaged over five sweeps by a signal averager. Type I (fig. 1A and fig. 2A, B, C) fibers ( 3 3 ~ of total fibers) discharged phasically during the inspiratory cycle of phrenic nerve when the animals breathed 100~o 02 (fig. 2A). As the inspired CO2 level rose (up to 10~o), the frequency of discharge during inspiration also rose (fig. 2B). With low oxygen (12~ O_~,88~o N2) breathing the Type I fibers also increased their discharge frequency progressively in a manner similar to high CO2 breathing but the rate of discharge per breath was higher than during CO2 breathing (fig. 2C). Type II fibers (45~o of the total) had phasic activity during inspiration but also had expiratory activity. They had a higher CO2 threshold than Type I fibers (fig.
60
J. MITRA AND N.S. CHERNIACK
m
TYPE I
B
A
]' L
D
H 3.0 sec.
G 100% 0 2
E
H 10% ETCO 2
[1 m C
TYPE II
l
TYPE III
I
FiO212%
Fig. 2. Records of single fiber activity during hypercapnia and hypoxia. The upper tracing in each frame is a record of Schmitt Trigger pulses of the hypoglossal single fiber activity; the lower tracings are integrated whole phrenic nerve activity. All tracings are average of five sweeps processed by a Signal Averager (see text). Spontaneous activity at 100K 02 breathing (end-tidal CO2 4.0~o) for Type I, II and III fibers are shown in frames A, D and G, respectively. The response of the same group of fibers during hypercapnia with an end-tidal CO2 of 10~ (10~o ET CO2) are shown in frames B, E and H. The effects of 12~o 02 breathing (F[o~ 12~) are shown in C, F and I. 1B, C, D, and 2D, E, F). D u r i n g C O 2 rebreathing Type II fibers discharged vigorously during the inspiratory phase at different levels o f elevated CO2 (fig. 2E). As the CO2 level rose, the discharge rate - - b o t h inspiratory phasic and expiratory activityincreased slowly. Type II fibers responded to hypoxia in a m a n n e r similar to hypercapnia (fig. 2F). Type I I I fibers ( 2 2 ~ o f total) had c o n t i n u o u s r a n d o m activity at 100~o 02 (fig. 1E) which increased gradually with increasing CO2 level and hypoxia (fig. 2G, H, I).
Effect of end-tidal C02 on discharge patterns of the three types of fibers.
A typical response o f the three types o f fibers to different levels o f hypercapnia is shown in fig. 3. At end-tidal (ET) CO2 levels o f 5 to 6~o Type I and II fibers showed little increase in discharge frequency. As the ET CO2 rose to 7 ~ the discharge frequency o f Type I increased faster than Type II. However, above ET CO2 7~o, both Type I and II increased their discharge rates in a similar fashion. The rate
SINGLE HYPOGLOSSALNERVE FIBER ACTIVITY
61
6O .
~
typeII
40
¢ 3o
.............~"".........,../.y,"""t'"'"'"'"'"'"'""
~¢r I
5
i
6
i
7 End Tidal CO 2
i
8 (%)
i
9
type III
I
10
Fig. 3. Effect of end-tidal CO2 on discharge patterns of the three types of fibers. Mean discharge frequency during inspiration of three types of hypoglossal nerve fibers (on ordinate) versus the percentage of end-tidal CO2 (on abscissa). The vertical lines indicate the standard eror of the mean (SEM) of ten successivebreaths of three individual fibers. In this and in subsequent figures the response patterns are typical for three groups of fibers.
of discharge of both Type I and II fibers increased disproportionately faster at high ET CO2 levels (above 7 ~ ) than at lower levels. On the other hand, Type III fibers increased their discharge rate approximately linearly over the whole range of CO2 rebreathing. Analysis of variance (F-test) of all 42 fibers showed that the difference in inspiratory discharge frequency among all three groups of fibers was highly significant (F <0.001) between ET 7~o and 9 ~ CO2. Above 9~o CO2 the difference between Type I and II was not significant. The activity of Type III was highly significantly different (F <0.001) above 7 ~ CO2 from both Type I and Type II fibers.
Inspiratory discharge frequency during hypercapnia as a function of integrated phrenic nerve activity. We also examined the relationship between the discharge frequency of the three types o f fibers and peak phrenic nerve activity. A typical response pattern is shown in fig. 4. The peak amplitude of the phrenic nerve activity was expressed as a percentage change from the activity during 100% 02 breathing. The discharge frequency of Type I and II fibers did not increase appreciably until the phrenic nerve activity rose from control levels by 20 and 30% respectively. On the other hand, the discharge frequency of Type III fibers increased almost linearly with increased phrenic activity. Analysis of variance showed that the difference in activity between Type I and II fibers are highly significant (F <0.001) up to 4 0 ~ of phrenic nerve amplitude. However, above 4 0 ~ of phrenic nerve amplitude the difference between the fiber
62
J. MITRA AND N. S. CHERNIACK 60
_=
. ..,"".I tYpeI ..y..y........~"~ typeII
40 c
==
g3o ,,= ='2o
.=o_ ~.
:E
...................... •
.......................
•
,,,
4o
I
I
I
I
I
10
20
30
40
50
% Change in P e a k Amplitude of the Integrated
Phrenic Nerve Activity
Fig. 4. Inspiratory dischargefrequencyof hypoglossal fibers vs peak amplitude of the integrated phrenic nerve activity during hypercapnia. Mean discharge frequency during inspiration of the three hypoglossal nerve fiber types (on ordinate) vs peak amplitude of the integrated phrenic nerve activity (on abscissa). Peak amplitude of the integrated phrenic nerve activity is expressed as percent change from the activity during control on 100~o02. The vertical bars are SEM of hypoglossal nerve fiber discharges of ten successive breaths of three individual fibers. groups I and II became insignificant. The 'F' value for Type III fibers with respect to Type I and II fibers were highly significant (F <0.001) above 3 0 ~ of the phrenic nerve amplitude and remained so throughout.
Inspiratory discharge frequency during hypoxia as a function of integrated peak phrenie activity. The relationship between the discharge rate of three types of fibers during 12~o 02 breathing was somewhat different than that of hypercapnia. A typical response is shown in fig. 5. As in the previous figure, the amplitude of the phrenic activity has been expressed as a percentage of the activity during 100~o O2 breathing. With the hypoxia produced by 12~ 02, the phrenic activity also increased. The discharge rate of the hypoglossal fibers also increased progressively. Whereas Type III fiber increased its activity linearly, Type I and II fibers increased their discharge frequency more curvilinearly during hypoxia. 'F' tests were highly significant ( F <0.001) for the activity of all three groups of fibers above 3 0 ~ of phrenic nerve amplitude.
Relationship between conduction velocities of different fiber sizes and their responses to hypercapnia and hypoxia. A total number of 26 fibers were used to measure conduction velocity. The conduction velocities of all fiber types were measured while the animal was breathing 100~ 02. There was an overlap in conduction velocity among the three types. However, in general, Type I had a mean conduction
SINGLE HYPOGLOSSAL NERVE FIBER ACTIVITY
63
60
,5
T ; type,
-~ 50
/'"'"
":" 40
type II
../..'"
type III
g2o
{
I
I
10
2O
I
I
I
:3O
4O
50
% Change in Peak Amplitude of the Integrated Phrenic Nerve Activity
Fig. 5. Inspiratory discharge frequency of hypoglossal nerve fibers during hypoxia as a function of peak amplitude o f the integrated phrenic nerve activity. Mean inspiratory discharge frequency of the three types of hypoglossal nerve fibers (on ordinate) v s the peak amplitude of the integrated phrenic nerve (on abscissa) expressed as a percentage change from 100~ 02 breathing. Vertical bars are SEM of hypoglossal nerve fiber discharges of five successive breaths of three individual fibers.
velocity of 30.0 + 6.2 (SE) m/see. Type II had a mean of 36.7 + 4.3 m/see. Type III had a mean of 41.3 + 5.1 m/see. The 'F' test showed that the differences among the three groups of fibers are highly significant (F <0.001).
Discussion
Our results are in agreement with the observations of others (Blom, 1960; Lei, 1961; Lowe and Sessel, 1973; Miller and Bowman, 1974) that in the cat and the monkey genioglossus muscle activity is in phase with inspiration. Sumi (1964) recorded from single hypoglossal fibers in decerebrate cats and found that spontaneous activity did not occur in these fibers until asphyxia was induced. However, we found a respiratory modulated discharge in some single hypoglossal nerve fibers (Type I) even during 100~o 02 breathing. This difference could be due to the difference in experimental preparations. However, in a subsequent study, Sumi (1967) found that in intact anesthetized kittens a few fibers exhibited a respiratory rhythm during normal breathing. Later, in intra- and extracellular studies of the hypoglossal motoneuron pool in decerebrate cats, the same investigator (Sumi, 1969) reported bursts of action potentials which coincided with some phase of respiration. Sumi did not investigate the response of these motoneuron pools to hypercapnia and hypoxia.
64
J. MITRAAND N.S. CHERNIACK
According to the anatomical findings of Rexed (1944) and others (Blom, 1960; Egel et al., 1968) the fibers in the main trunk of the hypoglossal nerve are unimodally distributed with the peak occurring in the vicinity of 7-9/~m in diameter. Our observations suggest that although the hypoglossal nerve trunk is composed of relatively homogeneous fiber sizes, these fibers show functional differences. On close inspection we could identify five different types of discharge patterns during inspiration and expiration. Some of these discharge patterns have been observed by Cohen (1970) in bulbopontine respirator3; neurons. According to Baumgarten et al. (1957) and Haber et al. (1957) the 'inspiratory' neurons are found predominantly in the medulla, rostral to the obex. In contrast, the 'inspiratory-expiratory' neurons are found predominantly in the rostral pons (Cohen and Wang, 1959). We are not aware of any work where the anatomical connection between respiratory related neurons of the pons and medulla and the hypoglossal motoneuron pool have been established. So at this time we can only speculate that the respiratory neurons of the pons and medulla may directly influence the activity of the hypoglossal motoneuron pool in order to synchronize tongue movement during respiration. On the basis of responses to hyperoxic hypercapnia it seems that different hypoglossal motoneurons have different CO2 activating thresholds. Our findings are consistent with the idea that Type I may be low and Type II high CO2 threshold fibers. We have not studied the effects of CO: levels over 10~. So, the response of Type III fiber under very high CO2 levels remains speculative. It is possible that Type III fibers are actually very high threshold fibers. The position of the anterior wall of the pharynx is determined at least in part by the genioglossus muscle. Our findings suggest that the Type I and Type II fibers may cause contraction of the genioglossus muscle during inspiration, which would protrude the tongue rhythmically in phase with breathing. On the other hand, the expiratory activity of Type II fiber and the continuous activity of Type III fiber may activate the genioglossus muscle to advance the base of the tongue and thus enlarge the volume of the oropharynx all through the breathing cycle. The combined activity of all three types of fibers during hypercapnia or hypoxia might reduce the resistance to airflow through the oropharynx. It also has been suggested (Lowe, 1981) that tongue movement in phase with respiration may play a role in the stabilization of the larynx. The hypoglossal nerve is also involved in positioning of the tongue during chewing, deglutition and vocalization. It is not clear from our study whether or not CO: influences the same motoneurons that are involved in tongue positioning. Nevertheless, it is interesting that a cranial nerve like the hypoglossal has fibers that are precisely time-locked with inspiratory cycle. From whole nerve recordings, it is known that other cranial nerves that supply the upper airway muscles (i.e., vagus, facial and trigeminal) (Cohen, 1979) also show respiratory rhythm. It would be interesting to investigate whether or not their motoneurons have responses similar to hypoglossal motoneurons during increased chemical drive. We observed that conduction velocities of three types of fiber overlap considera-
SINGLE HYPOGLOSSAL NERVE FIBER ACTIVITY
65
bly. In general; Type l~fiber had sIowest (30.0 m/sec) and Type III highest (41.3 m/sec) conduction velocity. Type II had conduction velocity that was in between the two types (36.7 m/sec). It appears that Type I fibers have their origin from small motoneurons. It is possible that the smaller motoneurons are more often inspiratory phase locked than the larger motoneurons. It is known that the tonic motoneurons of skeletal muscle are smaller and the phasic motoneurons are larger (Granit, 1972). It appears, that at least during hypercapnia and hypoxia, the hypoglossal motoneurons may behave differently than skeletal muscle motoneurons of other parts of the body (viz. larger motoneurons cause tonic activity of the tongue).
Acknowledgements This work was supported by NIH Program Project grant HL-25830 and V.A. Merit Review. References Andrew, B. L. (1955). The respiratory displacement of the larynx: a study of the innervation of accessory respiratory muscles. J. Physiol. (London) 130: 474-487. Baumgarten, R.V., A.V. Baumgarten and K.P. Schaefer (1957). Beitrag zur Lokalisationsfrage bulboreticul/i.rer respiratorischer Neurone der Katze. Pfliigers Arch. 264: 217-227. Blom, S. (1960). Afferent influences on tongue muscle activity. Acta Physiol. Scand. 49 (Suppl. 170): 1 97. Brouillette, R.T. and B.T. Thach (1980). Control of genioglossus muscle inspiratory activity. J. Appl. Physiol. 49:801 808. Cohen, M. 1. and S.C. Wang (1959). Respiratory neuronal activity in pons of cat. J. Neurophysiol. 22 : 33-50. Cohen, M. I. (1970). How respiratory rhythm originates: evidence from discharge pattern of brainstem respiratory neurons. In: Breathing. Hering-Breuer Centenary Symposium, edited by R. Porter. London, J. and A. Churchill, pp. 125-150. Cohen, M. I. (1979). Neurogenesis of respiratory rhythm in the mammal. Physiol. Rev. 59:1105 1173. Egel, R., J. P. Bowman and C.M. Combs (1968). Calibre spectra of the lingual and hypoglossal nerves of the rhesus monkey. J. Comp. Neurol. 134:163 174. Granit, R. (1972). Mechanisms Regulating the Discharge of Motoneurons. Springfield, IL, Charles C. Thomas, pp. 1-26. Haber, E., K. W. Kohn, S. H. Ngai, D. A. Holaday and S. C. Wang (1957). Localization of spontaneous respiratory neuronal activities in the medulla oblongata of the cat: A new location of the expiratory center. Am. J. Physiol. 190: 350-355. Lei, L. (1961). Irradiation of respiratory centre excitation to the motor centres for tongue muscles. Fiziol. Zh. S.S.S.R. 47: 906-912. Lowe, A.A. and B.J. Sessel (1973). Tongue activity during respiration, jaw opening, and swallowing in cat. Can. J. Physiol. Pharmacol. 51:1009-1011. Lowe, A. (1981). The neural regulation of tongue movements. Prog. Neurobiol. 15: 295-344. Miller, A.J. and J.P. Bowman (1974). Divergent synaptic influences affecting discharge patterning of genioglossus motor units. Brain Res. 78:179 191. Mitchinson, A. G. and J. M. Yoffey (1947). Respiratory displacement of larynx, hyoid bone and tongue. J. Anat. 81: 118-121.
66
J. MITRA A N D N. S. C H E R N I A C K
Onal, E., M. Lopata and T.D. O'Connor (1981). Diaphragmatic and genioglossal electromyogram responses to CO 2 rebreathing in humans. J. Appl. Physiol. 50: 1052-1055. Paintal, A. S. (1953). The conduction velocities of respiratory and cardiovascular afferent fibres in the vagus nerve. J. Physiol. (London) 121 : 341-359. Rexed, B. (1944). Contributions to the knowledge of the postnatal development of the peripheral nervous system in man. Acta I~sychiea. Nen'r. S.ra~d~. f~p~. ~ : 2~-2~06~ Sumi, T. (1964). Neuronal mechanisms in swallowing. Pfliigers Arch. 278: 467-477. Sumi, T. (1967). The nature and postnatal development of reflex diglutition in the kitten. Jpn. J. Physiol. 17: 200-210. Sumi, T. (1969). Functional differentiation of hypoglossal motoneurons in cats. Jpn. J. Physiol. 19: 55-67. Weiner, D., J. Mitra, J. Salamone and N.S. Cherniack (1980). The effect of chemical and reflex stimulation on phrenic, hypoglossal, and recurrent laryngeal nerve activity. Fed. Proc. 33: 322. Weiner, D., J. Mitra, J. Salamone and N.S. Cherniack (1982). Effect of chemical stimuli on nerves supplying upper airway muscles. J. Appl. Physiol. 52: 53~536.
55
Elsevier
T H E EFFECTS OF H Y P E R C A P N I A A N D H Y P O X I A O N S I N G L E H Y P O G L O S S A L N E R V E FIBER ACTIVITY
J Y O T I M I T R A and N E l L S. C H E R N I A C K Department of Medicine, Case Western Reserve University, Cleveland, OH 44106, U.S.A.
Abstract. The respiratory related modulation of hypoglossal nerve activity has been studied at the single fiber level in cats under hyperoxic hypercapnia and hypoxic conditions and their conduction velocities determined. Changes in fiber activity were compared to simultaneous changes occurring in phrenic activity. Three different kinds of discharge patterns were observed: (a) inspiratory, (b) phasic activity during both inspiration and expiration, and (c) continuous random activity with no respiratory modulation. These fibers could be grouped into three categories according to their pattern of discharge during CO 2 breathing. Type I fibers, mean conduction velocity of 30.0 m/sec, exhibited only an inspiratory phasic discharge during 100~o 02 breathing. Their discharge frequency increased rapidly with higher levels of CO 2 and hypoxia. Type II fibers, mean conduction velocity of 36.7 m/sec, had three different kinds of inspiratoryexpiratory discharge patterns during 100~ 02 breathing. With increasing hypercapnia or hypoxia fibers of this group discharged phasically during inspiration and discharged at low frequency during expiration. Type III fibers had a non phasic discharge pattern at 100~o 02 breathing and at all levels of CO 2 tested (up to 10~). Discharge frequency rose during CO 2 rebreathing and hypoxia, but the rate of increase was much less than Type I and Type II fibers. Their mean conduction velocity was 41.3 m/sec. The inspiratory activity of Type I and II fibers increased their activity more than the phrenic during hypercapnia and hypoxia. Type I1 and Type III fibers are responsible at least in part for the tonic activity of the nerve. Cat Control of breathing Hypercapnia Hyperoxia
Hypoglossal nerve Hypoxia Nerve fiber Phrenic nerve
Accepted Jbr publication 4 July 1983 Address correspondence to: Dr. Jyoti Mitra, Department of Medicine, University Hospitals, 2074 Abington Road, Cleveland, OH 44106, U.S.A. 0034-5687/83/$03.00 © 1983 Elsevier Science Publishers B.V.
56
J. MITRA AND N.S. CHERNIACK
The activity of many of the cranial nerves that supply the upper airway muscles is modulated by respiration. This activity appears to be especially important during sleep in maintaining upper airway patency (Brouillette and Thach, 1980). Loss of tonic and phasic activity in laryngeal muscles and oral muscles such as the genioglossus is believed to lead to obstruction during inspiration in some sleeping humans (Onal et al., 1981). The changes in genioglossus muscle activity that occur with respiration have been investigated in both animals and humans (Andrew, 1955; Blom, 1960; Brouillette and Thach, 1980; Lei, 1961; Lowe and Sessel, 1973; Mitchinson and Yoffey, 1947; Miller and Bowman, 1974; Onal et al., 1981). Studies from our laboratory (Weiner et al., 1980, 1982) have indicated that the integrated hypoglossal nerve activity (medial branch) - - the motor nerve to genioglossus muscle - - has both phasic and tonic activity during respiratory drive. While the tonic activity is linearly related to phrenic nerve activity, the phasic activity tends to increase their activity more than that of the phrenic nerve during hypercapnia and hypoxia. The purpose of our present investigation was two-fold : (1) to study whether the phasic and tonic activity of the integrated whole nerve could be related to discharge patterns occurring in a single fiber preparation; and (2) to determine how hypercapnia and hypoxia affects different single fibers.
Materials and methods
Acute experiments were performed on 15 cats of both sexes (1.54.5 kg body weight) which were anesthetized with alpha chloralose (36 mg/kg) administered intraperitoneally. Local anesthesia induced by 2 ~ Lidocaine (Xylocaine) was used over pressure points and in incised tissue. Body temperature was maintained at 38 _+ 1 °C using an electric thermal blanket. The saphenous vein of one forelimb was cannulated for infusion of saline and Flaxedil (4 mg/kg). The trachea was intubated for artificial ventilation and to allow the insertion of a capnograph (GodartStatham) probe to monitor end-tidal CO2. Through a midline incision, extending from the symphysis of the lower jaw to the manubrium, both the hypoglossal (medial branch) and the fifth root of the phrenic nerve were exposed ipsilaterally. They were then separated from the surrounding tissues and severed at their most peripheral point. The former was used for a single nerve fiber preparation and the latter for monitoring central respiratory activity. Both vagus nerves were also isolated and cut in order to eliminate stretch receptor influences on hypoglossal motoneurons. The cutaneous edges of the incision were then elevated to make a trough which was filled with warmed mineral oil. The oil temperature was maintained at body temperature with the aid of a small warm water circulating coil immersed in the pool. The conduction velocity of a single fiber was measured according to the method described by Paintal (1953) with slight modification. In short, the connective tissue
S I N G L E H Y P O G L O S S A L NERVE FIBER ACTIVITY
57
sheath of the hypoglossal nerve was split and desheathed to a length of 6 cm. The most central part of the desheathed nerve was placed on a pair of stainlesssteel stimulating electrodes for stimulation later. The peripheral end of the nerve was then divided longitudinally into several smaller strands using an iridectomy knife. Under microscopic control, using a pair of sharpened dissecting needles, one' of the smaller strands was further dissected and placed on a monopolar stainless-steel recording electrode of 200 #m in diameter. An indifferent electrode was placed in the neck muscle. The distance between the stimulating and the recording electrodes was 6 cm. The activity of the strand was amplified (Grass Instruments, AC Preamplifier, P51 l) and fed in parallel to one channel of a dualbeam oscilloscope (Tektronix, 5113), an audio amplifier and to a tape recorder (Hewlett-Packard, 3968A). The nerve was then stimulated with rectangular pulses delivered by a stimulator (Grass Instruments, $48) connceted to the stimulating electrodes via an isolation unit (Grass Instruments, PSIU 6). The action potential so recorded was compound in nature. The strand was subdivided further until a single action potential could be obtained by stimulation. The conduction distance of the action potential in the selected strand was measured with a pair of dividers from the cathode of the stimulating electrodes to the recording electrode. Spontaneous discharge of the fibers thus isolated were also used to study the effects of hypercapnia and hypoxia. The fifth cervical root of the phrenic nerve (ipsilateral to the hypoglossal nerve) was cut peripherally and desheathed. The desheated nerve was then placed on a pair of stainless-steel hook electrodes (200 #m in diameter), immersed in warm mineral oil at the animal's body temperature. The output of the electrode was amplified (Grass Instuments, AC Preamplifier, P511), rectified and processed by a Paynter filter (time constant 200 msec) to obtain a moving average signal of the phrenic nerve and thereby to assess central respiratory drive. The moving average signal was connected to the second channel of the dual-beam oscilloscope to monitor the temporal relationship of hypoglossal unit discharge to the phrenic signal. The moving average signal was also stored on the magnetic tape. Recordings were made after the animals breathed 100~o 02 for 10 min, and then under conditions of increased chemical drive, progressive hyperoxic hypercapnia, and isocapnic hypoxia. Progressive hyperoxic hypercapnia was produced in the following way. A mixture of 5~o CO2 and 95~ 02 was delivered to the inspiratory port of the ventilator via a 2 L Collins bag. Gas from the expiratory line was then returned to the bag and the resulting mixture was rebreathed. The end-tidal CO2 was continuously measured with an infrared CO2 analyzer (Godart-Statham), and recorded on magnetic tape. Hypoxia was produced by connecting the inlet port of the ventilator to a 5 L Collins bag containing 12~ 02 and 88~ N2. The expiratory port of the ventilator remained open to atmosphere. Data were analyzed from the magnetic tape in the following way. First, tape segments were identified with different CO2 levels. Then the integrated phrenic
58
J. MITRA AND N. S. CHERNIACK
neurogram was displayed in one channel of a signal averager (Princeton Applied Research, 4202) and the averager was triggered by the inspiratory cycle of the phrenic nerve at normal averaging mode. Hypoglossal single fiber activity was processed by a Schmitt trigger circuit in order to convert fiber activity into uniform pulses of fixed amplitude and duration. The output pulses of the Schmitt trigger were then fed into the second channel of the averager. In this way the temporal relationship between the two signals were obtained at different endtidal CO2 levels. Counting of discharge frequency during inspiratory phase was made possible with the use of a comparator circuit and a gate-operated digital pulse counter (Charles Ward Enterprises). The comparator was set in such a way that during inspiration when voltage of the integrated phrenic neurogram rose above the baseline activity the comparator was turned on and remained so during the rest of the inspiratory phase. At the onset of expiration the comparator was turned off. The output signal from the comparator was used to open (during inspiration) and close (during expiration) the gate of the counter. In this way the counter counted spike activity only during the inspiratory phase of the respiratory cycle. Mean discharge rate during inspiratory phase was calculated dividing the total number of spikes during inspiration by the inspiratory time (TI). For observation of single fiber activity pattern, it was displayed on the oscilloscope by intensifying beam with 'z' axis modulation. Statistical analyses (analysis of variance) were also done to test the significance of discharge rate among the three types of fibers.
Results
Fiber responses in cats breathing oxygen A total of forty-two fibers were used in the study. In general during 100% 02 breating (end-tidal (ET) CO2 4.0~o) relatively few fibers were found phasically active. However, a close inspection of discharge patterns with respect to integrated phrenic nerve activity at 100~o 02 breathing revealed a variety of discharge patterns. Some of the discharge patterns we observed were the following. (a) Inspiratory. Fiber activity was restricted only to the inspiratory phase of the respiratory cycle. Such fibers were silent during the expiratory phase (fig. 1A). (b) Inspiratory-Pause-Expiratory (Burst). Frequency of discharge increased during inspiratory phase. However, at the peak of inspiration there was a pause. As expiration started there was a short burst of activity. Such fibers did not have activity during the rest of the expiratory phase (fig. 1B). (c) Inspiratory-Pause-Expiratory. These fibers did not have burst activity during expiration. The activity increased during inspiration and at the peak of inspiration there was a pause followed by resumption of continuous activity at a lower rate during the expiratory phase (fig. 1C).
59
SINGLE HYPOGLOSSAL NERVE FIBER ACTIVITY
A
B
C
1.0sec.
D
E
Fig. 1. Different discharge patterns observed during oxygen breathing. The upper trace of each frame shows the raw hypoglossal single fiber activity displayed on an oscilloscope by 'Z' axis modulation of
its beam intensity. The lower tracing shows the integrated phrenic nerve activity. All tracings are single sweep. A. Activity only during inspiration. B. Inspiratory activity as in A, followed by a pause and then a short burst of activity during expiration. C. Inspiratory activity followed by a pause as in B, but activity was resumed at a lower rate during expiration. There was no burst activity during expiration. D. Higher activity during inspiration, followed by lower activity during expiration without any pause. E. Random activity not changing with inspiration or expiration.
(d) Continuous-Peak frequency in &spiration. These fibers had continuous activity through the respiratory cycle and had no pause at the peak of inspiration (compare with fig. 1C). However, the activity increased during inspiration and decreased during expiration (fig. 1D). (e) Continuous, no respiratory modulation. These fibers did not have respiratory modulation and the discharge patterns were continuous and random (fig. 1E).
Effects ofhypercapnia and hypoxia.
The single fibers could be divided into three types based on their responses to hypercapnia and hypoxia as shown in fig. 2. Figure 2 displays processed hypoglossal single fiber activity together with the integrated phrenic nerve activity averaged over five sweeps by a signal averager. Type I (fig. 1A and fig. 2A, B, C) fibers ( 3 3 ~ of total fibers) discharged phasically during the inspiratory cycle of phrenic nerve when the animals breathed 100~o 02 (fig. 2A). As the inspired CO2 level rose (up to 10~o), the frequency of discharge during inspiration also rose (fig. 2B). With low oxygen (12~ O_~,88~o N2) breathing the Type I fibers also increased their discharge frequency progressively in a manner similar to high CO2 breathing but the rate of discharge per breath was higher than during CO2 breathing (fig. 2C). Type II fibers (45~o of the total) had phasic activity during inspiration but also had expiratory activity. They had a higher CO2 threshold than Type I fibers (fig.
60
J. MITRA AND N.S. CHERNIACK
m
TYPE I
B
A
]' L
D
H 3.0 sec.
G 100% 0 2
E
H 10% ETCO 2
[1 m C
TYPE II
l
TYPE III
I
FiO212%
Fig. 2. Records of single fiber activity during hypercapnia and hypoxia. The upper tracing in each frame is a record of Schmitt Trigger pulses of the hypoglossal single fiber activity; the lower tracings are integrated whole phrenic nerve activity. All tracings are average of five sweeps processed by a Signal Averager (see text). Spontaneous activity at 100K 02 breathing (end-tidal CO2 4.0~o) for Type I, II and III fibers are shown in frames A, D and G, respectively. The response of the same group of fibers during hypercapnia with an end-tidal CO2 of 10~ (10~o ET CO2) are shown in frames B, E and H. The effects of 12~o 02 breathing (F[o~ 12~) are shown in C, F and I. 1B, C, D, and 2D, E, F). D u r i n g C O 2 rebreathing Type II fibers discharged vigorously during the inspiratory phase at different levels o f elevated CO2 (fig. 2E). As the CO2 level rose, the discharge rate - - b o t h inspiratory phasic and expiratory activityincreased slowly. Type II fibers responded to hypoxia in a m a n n e r similar to hypercapnia (fig. 2F). Type I I I fibers ( 2 2 ~ o f total) had c o n t i n u o u s r a n d o m activity at 100~o 02 (fig. 1E) which increased gradually with increasing CO2 level and hypoxia (fig. 2G, H, I).
Effect of end-tidal C02 on discharge patterns of the three types of fibers.
A typical response o f the three types o f fibers to different levels o f hypercapnia is shown in fig. 3. At end-tidal (ET) CO2 levels o f 5 to 6~o Type I and II fibers showed little increase in discharge frequency. As the ET CO2 rose to 7 ~ the discharge frequency o f Type I increased faster than Type II. However, above ET CO2 7~o, both Type I and II increased their discharge rates in a similar fashion. The rate
SINGLE HYPOGLOSSALNERVE FIBER ACTIVITY
61
6O .
~
typeII
40
¢ 3o
.............~"".........,../.y,"""t'"'"'"'"'"'"'""
~¢r I
5
i
6
i
7 End Tidal CO 2
i
8 (%)
i
9
type III
I
10
Fig. 3. Effect of end-tidal CO2 on discharge patterns of the three types of fibers. Mean discharge frequency during inspiration of three types of hypoglossal nerve fibers (on ordinate) versus the percentage of end-tidal CO2 (on abscissa). The vertical lines indicate the standard eror of the mean (SEM) of ten successivebreaths of three individual fibers. In this and in subsequent figures the response patterns are typical for three groups of fibers.
of discharge of both Type I and II fibers increased disproportionately faster at high ET CO2 levels (above 7 ~ ) than at lower levels. On the other hand, Type III fibers increased their discharge rate approximately linearly over the whole range of CO2 rebreathing. Analysis of variance (F-test) of all 42 fibers showed that the difference in inspiratory discharge frequency among all three groups of fibers was highly significant (F <0.001) between ET 7~o and 9 ~ CO2. Above 9~o CO2 the difference between Type I and II was not significant. The activity of Type III was highly significantly different (F <0.001) above 7 ~ CO2 from both Type I and Type II fibers.
Inspiratory discharge frequency during hypercapnia as a function of integrated phrenic nerve activity. We also examined the relationship between the discharge frequency of the three types o f fibers and peak phrenic nerve activity. A typical response pattern is shown in fig. 4. The peak amplitude of the phrenic nerve activity was expressed as a percentage change from the activity during 100% 02 breathing. The discharge frequency of Type I and II fibers did not increase appreciably until the phrenic nerve activity rose from control levels by 20 and 30% respectively. On the other hand, the discharge frequency of Type III fibers increased almost linearly with increased phrenic activity. Analysis of variance showed that the difference in activity between Type I and II fibers are highly significant (F <0.001) up to 4 0 ~ of phrenic nerve amplitude. However, above 4 0 ~ of phrenic nerve amplitude the difference between the fiber
62
J. MITRA AND N. S. CHERNIACK 60
_=
. ..,"".I tYpeI ..y..y........~"~ typeII
40 c
==
g3o ,,= ='2o
.=o_ ~.
:E
...................... •
.......................
•
,,,
4o
I
I
I
I
I
10
20
30
40
50
% Change in P e a k Amplitude of the Integrated
Phrenic Nerve Activity
Fig. 4. Inspiratory dischargefrequencyof hypoglossal fibers vs peak amplitude of the integrated phrenic nerve activity during hypercapnia. Mean discharge frequency during inspiration of the three hypoglossal nerve fiber types (on ordinate) vs peak amplitude of the integrated phrenic nerve activity (on abscissa). Peak amplitude of the integrated phrenic nerve activity is expressed as percent change from the activity during control on 100~o02. The vertical bars are SEM of hypoglossal nerve fiber discharges of ten successive breaths of three individual fibers. groups I and II became insignificant. The 'F' value for Type III fibers with respect to Type I and II fibers were highly significant (F <0.001) above 3 0 ~ of the phrenic nerve amplitude and remained so throughout.
Inspiratory discharge frequency during hypoxia as a function of integrated peak phrenie activity. The relationship between the discharge rate of three types of fibers during 12~o 02 breathing was somewhat different than that of hypercapnia. A typical response is shown in fig. 5. As in the previous figure, the amplitude of the phrenic activity has been expressed as a percentage of the activity during 100~o O2 breathing. With the hypoxia produced by 12~ 02, the phrenic activity also increased. The discharge rate of the hypoglossal fibers also increased progressively. Whereas Type III fiber increased its activity linearly, Type I and II fibers increased their discharge frequency more curvilinearly during hypoxia. 'F' tests were highly significant ( F <0.001) for the activity of all three groups of fibers above 3 0 ~ of phrenic nerve amplitude.
Relationship between conduction velocities of different fiber sizes and their responses to hypercapnia and hypoxia. A total number of 26 fibers were used to measure conduction velocity. The conduction velocities of all fiber types were measured while the animal was breathing 100~ 02. There was an overlap in conduction velocity among the three types. However, in general, Type I had a mean conduction
SINGLE HYPOGLOSSAL NERVE FIBER ACTIVITY
63
60
,5
T ; type,
-~ 50
/'"'"
":" 40
type II
../..'"
type III
g2o
{
I
I
10
2O
I
I
I
:3O
4O
50
% Change in Peak Amplitude of the Integrated Phrenic Nerve Activity
Fig. 5. Inspiratory discharge frequency of hypoglossal nerve fibers during hypoxia as a function of peak amplitude o f the integrated phrenic nerve activity. Mean inspiratory discharge frequency of the three types of hypoglossal nerve fibers (on ordinate) v s the peak amplitude of the integrated phrenic nerve (on abscissa) expressed as a percentage change from 100~ 02 breathing. Vertical bars are SEM of hypoglossal nerve fiber discharges of five successive breaths of three individual fibers.
velocity of 30.0 + 6.2 (SE) m/see. Type II had a mean of 36.7 + 4.3 m/see. Type III had a mean of 41.3 + 5.1 m/see. The 'F' test showed that the differences among the three groups of fibers are highly significant (F <0.001).
Discussion
Our results are in agreement with the observations of others (Blom, 1960; Lei, 1961; Lowe and Sessel, 1973; Miller and Bowman, 1974) that in the cat and the monkey genioglossus muscle activity is in phase with inspiration. Sumi (1964) recorded from single hypoglossal fibers in decerebrate cats and found that spontaneous activity did not occur in these fibers until asphyxia was induced. However, we found a respiratory modulated discharge in some single hypoglossal nerve fibers (Type I) even during 100~o 02 breathing. This difference could be due to the difference in experimental preparations. However, in a subsequent study, Sumi (1967) found that in intact anesthetized kittens a few fibers exhibited a respiratory rhythm during normal breathing. Later, in intra- and extracellular studies of the hypoglossal motoneuron pool in decerebrate cats, the same investigator (Sumi, 1969) reported bursts of action potentials which coincided with some phase of respiration. Sumi did not investigate the response of these motoneuron pools to hypercapnia and hypoxia.
64
J. MITRAAND N.S. CHERNIACK
According to the anatomical findings of Rexed (1944) and others (Blom, 1960; Egel et al., 1968) the fibers in the main trunk of the hypoglossal nerve are unimodally distributed with the peak occurring in the vicinity of 7-9/~m in diameter. Our observations suggest that although the hypoglossal nerve trunk is composed of relatively homogeneous fiber sizes, these fibers show functional differences. On close inspection we could identify five different types of discharge patterns during inspiration and expiration. Some of these discharge patterns have been observed by Cohen (1970) in bulbopontine respirator3; neurons. According to Baumgarten et al. (1957) and Haber et al. (1957) the 'inspiratory' neurons are found predominantly in the medulla, rostral to the obex. In contrast, the 'inspiratory-expiratory' neurons are found predominantly in the rostral pons (Cohen and Wang, 1959). We are not aware of any work where the anatomical connection between respiratory related neurons of the pons and medulla and the hypoglossal motoneuron pool have been established. So at this time we can only speculate that the respiratory neurons of the pons and medulla may directly influence the activity of the hypoglossal motoneuron pool in order to synchronize tongue movement during respiration. On the basis of responses to hyperoxic hypercapnia it seems that different hypoglossal motoneurons have different CO2 activating thresholds. Our findings are consistent with the idea that Type I may be low and Type II high CO2 threshold fibers. We have not studied the effects of CO: levels over 10~. So, the response of Type III fiber under very high CO2 levels remains speculative. It is possible that Type III fibers are actually very high threshold fibers. The position of the anterior wall of the pharynx is determined at least in part by the genioglossus muscle. Our findings suggest that the Type I and Type II fibers may cause contraction of the genioglossus muscle during inspiration, which would protrude the tongue rhythmically in phase with breathing. On the other hand, the expiratory activity of Type II fiber and the continuous activity of Type III fiber may activate the genioglossus muscle to advance the base of the tongue and thus enlarge the volume of the oropharynx all through the breathing cycle. The combined activity of all three types of fibers during hypercapnia or hypoxia might reduce the resistance to airflow through the oropharynx. It also has been suggested (Lowe, 1981) that tongue movement in phase with respiration may play a role in the stabilization of the larynx. The hypoglossal nerve is also involved in positioning of the tongue during chewing, deglutition and vocalization. It is not clear from our study whether or not CO: influences the same motoneurons that are involved in tongue positioning. Nevertheless, it is interesting that a cranial nerve like the hypoglossal has fibers that are precisely time-locked with inspiratory cycle. From whole nerve recordings, it is known that other cranial nerves that supply the upper airway muscles (i.e., vagus, facial and trigeminal) (Cohen, 1979) also show respiratory rhythm. It would be interesting to investigate whether or not their motoneurons have responses similar to hypoglossal motoneurons during increased chemical drive. We observed that conduction velocities of three types of fiber overlap considera-
SINGLE HYPOGLOSSAL NERVE FIBER ACTIVITY
65
bly. In general; Type l~fiber had sIowest (30.0 m/sec) and Type III highest (41.3 m/sec) conduction velocity. Type II had conduction velocity that was in between the two types (36.7 m/sec). It appears that Type I fibers have their origin from small motoneurons. It is possible that the smaller motoneurons are more often inspiratory phase locked than the larger motoneurons. It is known that the tonic motoneurons of skeletal muscle are smaller and the phasic motoneurons are larger (Granit, 1972). It appears, that at least during hypercapnia and hypoxia, the hypoglossal motoneurons may behave differently than skeletal muscle motoneurons of other parts of the body (viz. larger motoneurons cause tonic activity of the tongue).
Acknowledgements This work was supported by NIH Program Project grant HL-25830 and V.A. Merit Review. References Andrew, B. L. (1955). The respiratory displacement of the larynx: a study of the innervation of accessory respiratory muscles. J. Physiol. (London) 130: 474-487. Baumgarten, R.V., A.V. Baumgarten and K.P. Schaefer (1957). Beitrag zur Lokalisationsfrage bulboreticul/i.rer respiratorischer Neurone der Katze. Pfliigers Arch. 264: 217-227. Blom, S. (1960). Afferent influences on tongue muscle activity. Acta Physiol. Scand. 49 (Suppl. 170): 1 97. Brouillette, R.T. and B.T. Thach (1980). Control of genioglossus muscle inspiratory activity. J. Appl. Physiol. 49:801 808. Cohen, M. 1. and S.C. Wang (1959). Respiratory neuronal activity in pons of cat. J. Neurophysiol. 22 : 33-50. Cohen, M. I. (1970). How respiratory rhythm originates: evidence from discharge pattern of brainstem respiratory neurons. In: Breathing. Hering-Breuer Centenary Symposium, edited by R. Porter. London, J. and A. Churchill, pp. 125-150. Cohen, M. I. (1979). Neurogenesis of respiratory rhythm in the mammal. Physiol. Rev. 59:1105 1173. Egel, R., J. P. Bowman and C.M. Combs (1968). Calibre spectra of the lingual and hypoglossal nerves of the rhesus monkey. J. Comp. Neurol. 134:163 174. Granit, R. (1972). Mechanisms Regulating the Discharge of Motoneurons. Springfield, IL, Charles C. Thomas, pp. 1-26. Haber, E., K. W. Kohn, S. H. Ngai, D. A. Holaday and S. C. Wang (1957). Localization of spontaneous respiratory neuronal activities in the medulla oblongata of the cat: A new location of the expiratory center. Am. J. Physiol. 190: 350-355. Lei, L. (1961). Irradiation of respiratory centre excitation to the motor centres for tongue muscles. Fiziol. Zh. S.S.S.R. 47: 906-912. Lowe, A.A. and B.J. Sessel (1973). Tongue activity during respiration, jaw opening, and swallowing in cat. Can. J. Physiol. Pharmacol. 51:1009-1011. Lowe, A. (1981). The neural regulation of tongue movements. Prog. Neurobiol. 15: 295-344. Miller, A.J. and J.P. Bowman (1974). Divergent synaptic influences affecting discharge patterning of genioglossus motor units. Brain Res. 78:179 191. Mitchinson, A. G. and J. M. Yoffey (1947). Respiratory displacement of larynx, hyoid bone and tongue. J. Anat. 81: 118-121.
66
J. MITRA A N D N. S. C H E R N I A C K
Onal, E., M. Lopata and T.D. O'Connor (1981). Diaphragmatic and genioglossal electromyogram responses to CO 2 rebreathing in humans. J. Appl. Physiol. 50: 1052-1055. Paintal, A. S. (1953). The conduction velocities of respiratory and cardiovascular afferent fibres in the vagus nerve. J. Physiol. (London) 121 : 341-359. Rexed, B. (1944). Contributions to the knowledge of the postnatal development of the peripheral nervous system in man. Acta I~sychiea. Nen'r. S.ra~d~. f~p~. ~ : 2~-2~06~ Sumi, T. (1964). Neuronal mechanisms in swallowing. Pfliigers Arch. 278: 467-477. Sumi, T. (1967). The nature and postnatal development of reflex diglutition in the kitten. Jpn. J. Physiol. 17: 200-210. Sumi, T. (1969). Functional differentiation of hypoglossal motoneurons in cats. Jpn. J. Physiol. 19: 55-67. Weiner, D., J. Mitra, J. Salamone and N.S. Cherniack (1980). The effect of chemical and reflex stimulation on phrenic, hypoglossal, and recurrent laryngeal nerve activity. Fed. Proc. 33: 322. Weiner, D., J. Mitra, J. Salamone and N.S. Cherniack (1982). Effect of chemical stimuli on nerves supplying upper airway muscles. J. Appl. Physiol. 52: 53~536.