Inhibitory influences on hypoglossal neural activity by stimulation of midpontine dorsal tegmentum in decerebrate cat

Brain Research, 479 (1989) 185-189 Elsevier

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Inhibitory influences on hypoglossal neural activity by stimulation of midpontine dorsal tegmentum in decerebrate cat Koichi Kawahara, Yoshimi Nakazono, Shigeru Kumagai, Yoshiko Yamauchi and Yoshimi Miyamoto Department of Information Engineering, Yamagata University, Yonezawa (Japan) (Accel,ted 11 October 1988)

Key words: Dorsal tegmental field; Diaphragm; Hypoglossalnerve; Mesencephalic cat

In the spontaneously breathing decerebrate cat, the properties of the suppressive effects en hypoglossal nerve activity and on diaphragmatic activity elicited by stimulation of the midpontine dorsal tegmentum (DTF area) were analyzed. Stimulation simultaneously decreased the activities of the hypoglossal nerve as well as that of the diaphragm. However, the inhibitory influences on the above two kinds of activities were different in nature. Diaphragmatic activity, once suppressed by stimulation, recovered and gradually became greater in amplitude in spite of the continuation of stimulation. In contrast, DTF stimulation depressed tonic discharges of the hypoglossal nerve, and the decreased tonic nerve activity persisted after stimulation ended. Rhythmic hypogiossal activity, once suppressed by stimulation, reappeared during DTF stimulation. Such a rhythmic ,zctivity,however, vanished after the termination of stimulation, although the rhythmic diaphragmatic activity did not. In the acute precollicular-postmammillary decerebrate cat (mesencephalic cat), stimulation of the dorsal part of the caudal tegmental field (DTF) in the pons along the midline results in long-lasting suppression of postural muscle tone 6's'9. Recently, we have found that DTF stimulation simultaneously suppressed the rhythmic diaphragmatic activity in addition to tonic discharges of the hindlimb extensor muscles 7. In some animals, stimulation results in apnea for 20 s or more. The coexistence of postural atonia and apnea elicited by DTF stimulation leads us to believe that such a preparation may enable one to analyze the pontine neuronal structures responsible for the regulation of respiration during REM sleep since postural atonia is considered to be one of the most prominent features of R E M sleep 4. The genioglossus muscle innervated by the h~tpoglossal nerve is the only muscle which causes the tongue to protrude and bilateral loss of its action leads to a relapse of the tongue. In awake human subjects, the genioglossi are activated during respiration, particularly during the inspiratory phase n. Sau-

erland and Harper 1° reported that the activity of the genioglossus muscle is depressed during R E M sleep in human subjects. Recent attention has been focused on the regulatory mechanisms of genioglossus muscle activity during REM sleep in connection with obstructive sleep apnea. In the present study, we ha,,e attempted to clarify the following: (1) Does DTF stimulation suppress not only diaphragmatic activity but also the activity of the hypoglossal nerve innervating the genioglossus muscle? (2) If the answer is affirmative, are there any differences in the DTF-elicited suppressive effects on the above two kinds of activities? Experimental procedures have been described elsewhere in detail 5'7'9. In short, 6 adult cats weighing 2.6-4.2 kg were surgically decerebrated at the precollicular-postmammillary level under halothane anesthesia. The head of the animal was fixed in a stereotaxic frame and the limbs were placed on a still treadmill. Electromyograms (EMGg) were recorded by implanting bipolar electrodes made of thin (70 /zm) copper wires into the bilateral soleus muscles

Correspondence: K. Kawahara, Department of Information Engir,eering, Yamagata University, Yonezawa 992, Japan. 0006-8993/89/$03.50© 1989Elsevier Science Publishers B.V. (Biomedical Division)

186 s) and recorded on an FM data recorder together with the soleus EMGs, CO2 tension of expired air and stimulating pulses. In some of the animals, correlation analysis was performed off-line with a personal computer (NEC PC-9801). This analysis enabled us to detect a rhythmic component of the recorded neural discharges. Signals were gated to eliminate base-line noise 3. Autocorrelation of hypoglos~a! nerve activity revealed the mean oscillation frequen,.7, a,tocorrelation of the diaphragmatic activity reveale~ tl,e mean respiratory frequency. Cross-correlogram~ between the diaphragmatic and hypoglossal nerve activities were also calculated. At the end of each experiment, the at~hnals were deeply anesthetized (pentobarbital sodium, i.v.) and

and the diaphragm. Bipolar recording of the hypoglossal nerve activity was performed by inserting thin (70 pm) copper wires completely insulated except at the tip. The peripheral end of the nerve was then ligated and sectioned. In this study, the diaphragmatic EMG was used to assess th ~ effects on respiration by stimulation of the brainstem. Phrenic nerve activity was not used for the purpose since sectioning of the peripheral end of the nerve to record efferent activity only would result in changes in the respiratory movements. We did not record the genioglossus E M G because of the bilateral ligation of the carotid arteries prior to decerebration. End-tidal p C O 2 was monitored with an infrared gas analyzer and recorded. The diaphragmatic E M G and hypoglossal nerve activity were then R-C integrated (time constant = 0.1

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Fig. 1. DTF-eliciteddepressive effects on diaphragmatic, hypoglossaland soleus muscle activities. A-C: are the results obtained from I ! a single animal. Stimulus intensity was about 40/~A. DTF stimulation decreased the tonic discharges ot the hypog,os~a, ne~e as well as that of the bilateral soleus muscles. The horizontal broken line in the record of INT HYPO represents an approximate tonic: activity level before the start of stimulation. Stimulation also decreased the respira+oryfrequency (A). Correlation analysis disclosed that tae tonic-like dis~arges of the hypoglossalnerve before the onset of stimulation were rhythmicallymodulated synchronized with the n:spixntory rhythm (B). During the mid to latter part of the stimulation, the rhythmic component of the hypoglossal discharges was synchronized wi;h the respiratory rhythm as well (C). DIA, diaphragmatic EMG; HYPO, hypoglossal nerve activity; INT HYPO, integrated hypo~iossal nerve activity; SOL EMG, EMG of soleus muscles; L, left side; R, fight side; pCO2, CO2 tension of expired air; DTF, dorsa, part of the midpontine tegmental field; DIA ACG, autocorrelogram of diaphragmatic activity; HYPO ACG, autoconeIogram of hypoglossal activity; D-H CCG, cross correlogram between diaphragmatic and hypogiossal activities.

187 then stimulating sites were marked with an electrolyric lesion (DC current of 20 p A , 20 s). The brain was fixed in 10% formalin for later histological examination. The locations of the electrode tips were determined with reference to the stereotaxic atlas of Snider and Niemer 12. In all of the animals tested, stimulation of the midline D T F ehcited simultaneous suppression of posrural tone a;-d respiration. Stimulation also depressed hypog=assal nerve activity. Fig. 1 shows an example of the re;ults. Prior to DTF stimulation, both the bilateral soleus muscles and the hypoglossal nerve exhibited almost tonic discharges. In contrast, the diaphragm showed rhythmic bursting discharges with a mean interval of 1.3 s (Fig. 1A). Correlation analysis was then performed on hypoglossal nerve activity to detect a rhythmic component in the nerve activity, if present at all. The tonic-like activity of the hypoglossal nerve before the onset of DTF stimulation was shown to be

rhythmically modulated (Fig. 1B, HYPO ACG). The mean interval of such a rhythm was 1.3 s and the rhythm was almost synchronized with the respiratory rhythm (Fig. 1B, D-H CCG). DTF stimulation decreased the respiratory frequency and tonic discharges of the hypoglossal nerve as well as of the bilateral soleus muscles. End-tidal pCO2 increased slightly because of the depression of respiratory movement. Although the DTF-elicited decrease in respiratory frequency persisted during the entire DTF stimulation period, the ampfitude of the rhythmic diaphragmatic activity gradually became greater. As a consequence, in this animal, endtidal pCO2 at the latter part of the stimulation was a slightly lower level (hypocapnia) compared with that before stimulation. Out of 6 animals tested, 2 animals showed similar changes in the pCO 2. In 3 animals, end-tidalpCO 2 remained at the same level as that before stimulation. In the remaining one, end-tidal p C O 2 was at a slightly higher level. These results sug-

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Fig. 2. Rhythmic hypoglossai and diaphragmatic activities observed during the DTF stimulation. A-C are the results obtained from the same animal as in Fig. 1. StimuLusintensitywas increased to 60 pA. The period of stimulation is indicated by a bold solid 5he under the lowest rec'~rd of the left soleus muscle EMG (A). Expanded sweeps of the records indicated by broken lines under the iategrated diaphragmatic ~nd hypoglossal activities in A are shown in B. It was evident that the rhythmic hypoglossal activityappearing during stimulation was ~dmostsynchronized with diaphragmatic activitywithout correlation analysis The stimt~us site was the same as that in Fig. 1 and is indkated by a black star in the schematic drawing of the cat's brain stem (C). INT DIA, inLegrateddiaphragmatic EMG; INT HYPO, integ:ated hypoglossal nerve activity. Other abbreviations are the same as those in Fig. 1. See text for details.

188 gest that tidal volume, which was not measured in this study, increased despite the decrease in respiratory frequency during DTF stimulation. Correlation analysis between the diaphragmatic and hypoglossal nerve activities was again performed during the latter part of stimulation (Fig. 1C). The mean respiratory interval was elongated to about 3.4 s (DIA ACG). The autocorrelogram of hypoglossal nerve activity (HYPO ACG) showed that there existed a rhythmic component in the tonic-like activity of the nerve. The rhythm of the hypoglossal nerve was synchronized with the respiratory rhythm (D-H CCG). The DTF-elicited suppressive effects on the tonic discharges of the bilateral soleus muscles and of ~he hypoglossal nerve persisted even after termination of stimulation (Fig. 1A, INT HYPO). Fig. 2A shows another example of the results obtained from the same cat as in Fig. 1. The stimulus intensity was increased from 40 ~A (Fig. 1) to 60 ~ A (Fig. 2). Before DTF stimulation, although the rhythmic component of hypoglossal nerve activity was not clear in the integrated hypoglossal nerve activity (INT HYPO), correlation analysis disclosed that the tonic-like discharges of the nerve were rhythmically modulated synchronized with the respiratory rhythm. DTF stimulation decreased the tonic and rhythmic discharges of the hypoglossal nerve. However, the nerve again began to show rhythmic burstL,lg discharges about 17 s after the onset of stimulation and the rhythm was almost synchronized with that of the diaphragmatic activity (Fig. 2B). The suppressive effects on the tonic discharges of the nerve persisted even after termination of stimulation, similar to the DTF-elicited suppression of soleus muscle activity. The rhythmic discharges of the nerve disappeared after stimulation. The stimulation site was the same as in Fig. 1 (P5.0, LR0, H-4.8) and is indicated by a black star in the schematic drawing of the cat's brainstem (Fig. 2C). The present study has demonstrated that DTF stimulation resulted in the depression of hypoglossal nerve activity, especially of the tonic discharges of the nerve, and that such a depressive effect on tonic discharges persisted even after termination of stimulation. A previous study 7 and this study have shown that DTF stimulation suppresses both postural muscle ~one and diaphragmatic activity. This DTF-elicited suppression of postural ~one (postural atonia)

and hypoglossal nerve activity is similar to that of the features observed during REM sleep. It is well known that postural atonia occurs during REM sleep 4. In addition, the activity of the genioglossus muscle innervated by the hypoglossal nerve is depressed during REM sleep ~°. Therefore, the decerebrate preparation described here may be an appropriate animal model for analyzing the brain stem neuronal structures responsible for the regulation of respiration during sleep. Sauerland and Mitchell n reported that the tonic activity of the genioglossus muscle reflects the efforts of the muscle to counteract the relapse of the tongue due to its own gravity. Simultaneous suppression of the phrenic and hypoglossal activities evoked by DTF stimulation seems very interesting from the point of view of the genesis of central as well as obstructive sleep apnea. Diaphragmatic activity, once sup,pre~ed by DTF stimulation, resumed and gradually became greater in amplitude in spite of the continuation of stimulation. We have previously demonstrated that the DTF-elicited suppressive effects on diaphragmatic activity persisted during the entire period of stimulation 7. Thus, the strong respiratory drives to overcome the exerted inhibitory influences must be brought about during DTF stimulation. We suggested that the most probable origin of such respiratory drives is the chemical changes in the arterial blood, emerging as a consequence of the accumulation of carbon dioxide and/or the lack of oxygen 7. In this study, rhythmic hypoglossal activity, once st:~:,pressed by DTF stimulation, reappeared almost in parallel with diaphragmatic activity (Fig. 2A). Bruce et al. 2 suggested that the COn-related drive inputs from central and per~,heral chemoreceptors to phrenic and hypoglossa~ motoneuron p~Jols are different. Brouillette and Thach 1 proposed t;lat hypoxia preferentially activates the genioglossal m'.~scle relative to the diaphragm. Since the bilateral c~:rotid arteries were ligated prior to decerebration in the preparations used here, the function of the peripheral chemoreceptors (bilateral carotid bodies~ ~vas likely to be reduced or abolished. Recovery of the diaphragmatic and hypoglossal rhythmic activities under the inhibitory influences elicited by DTF stimulation suggest that strong respiratory drives, probably of central chemoreceptor origin, both to the phrenic and hypoglossal motoneuron pools, ma~, be brought

189 about during D T F stimulation. W e express sincere appreciation to Prof. Y. Honda, Faculty of Medicine, Chiba University for his

1 Brouillette, R.T. and Thach, B.T., Control of genioglossal inspiratory activity, I. Appi. Physiol. Respir. Environ. Exerc. Physiol., 49 (1980) 801-808. 2 Bruce, E.N., Mitra, 1. and Cherniaek, N.S., Central and peripheral chemoreceptor inputs to phrenic and hypoglossal motoneurons, J Appl. Physiol. Respir. Er:viron. Exercise Physiol., 53 (1982) 1504-1511. 3 Iscoe, S., Respirator~ and stepping frequencies in conscious exercising cats, J. Appl. Physiol. Respir. Environ. Exerc. Physiol., 51 (1981)835-839. 4 Jouvet, M., Neurophysiology of the state of sleep, Physiol. Rev., 47 (1967) 117-177. 5 Kawahara, K., Kumagai, S., Nakazono, Y. and Miyamoto, Y., Analysis of entrainment of respiratory rhythm by somatic afferent stimulation in cats using phase response curves, Biol. Cybern., 58 (1988) 235-242. 6 Kawahara, K., Mt~ri, S., Tomiyama, T. and Kanaya, T., Discharges of neurons in the midpontine dorsal tegmentum of mesencephalic cat during locomotion, Brain Research, 341 (1985) 377-380. 7 Kawahara, Ko, Nakazono, Y., Kumagai, S., Yamauchi, Y.

helpful discussions of the results. We also thank Dr. K. O h w a d a , Laboratory of Animal Center, Faculty of Medicine, Y a m a g a t a University for preparing the, experimental animals.

and Miyamoto, Y., Parallel suppression of extensor muscle tone and respiration by stimulation of pontine dorsal tegmentum in decerebrate cat, Brain Research, in press. 8 Mori, S., Aoki, M., Kawahara, K. and Sakamoto, T., Level setting of posturai muscle tone and initiation of locomotion by MLR stimulation, Adv. Physiol. Sci., 1 (1981) 179-182. 9 Mori, S., Kawahara, K., Sakamoto, T., Aoki, M. and Tomiyama, T., Setting and resetting of level of postural muscle tone in decerebrate cat by stimulation of brain stem,/. Neurophysiol., 48 (1982) 737-748. 10 Sauerland, E.K. and Harper, R.M., The human tongue during sleep: electromyographic activity of the genioglossus muscle, Exp. Neurol., 51 (1976) 160-170. 11 Sauerland, E.K. and Mitchell, S.P., Electromyograp~c activity of the human genioglossus muscle in response tc res~ piration and to positional changes of the head, 2:~!}. Los Angeles Neurol., 35 (1970) 69-73. 12 Snider, R.S. and Niemer, W.T.A., Stereotaxic Atlas of the Cat Brain, University of Chicago Press, Chicago, 1961.