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Cardio-inhibitory mechanism in the gigantocellular reticular nucleus of the medulla oblongata
Brain Research, 178 (1979) 221-232
221
© Elsevier/North-HollandBiomedicalPress
CARDIO-INHIBITORY MECHANISM IN THE GIGANTOCELLULAR RETICULAR NUCLEUS OF THE MEDULLA OBLONGATA
J. S. KUO, Y. HWA and C. Y. CHAI Department of Physiology and Biophysics, National Defense Medical Center, and Kohlberg Memorial Medical ResearchLaboratory, Veterans GeneralHospital, Taipei (Taiwan)
(Accepted April 5th, 1979) Key words:
cardio-inhibitory mechanism - - gigantocellular reticular nucleus - bradycardia - - vagus nerve
SUMMARY A cardio-inhibitory mechanism was localized in the ventral part of the medullary gigantocellular reticular nucleus (GRN) in chloralose-urethane anesthetized cats. Stimulation of this mechanism produced an average 58.9 ~ reduction of the heart rate (calculated from 55 responsive points having more than 4 0 ~ reduction) associated mostly with hypotension, or no change or occasionally a slight increase of the arterial blood pressure. Midcollicular decerebration did not affect this bradycardia. The bradycardia following G R N stimulation of either side by a pair of symmetrically placed electrodes was reduced slightly but equally by: (1) sectioning either side of the vagus nerve; (2) hemisection at a level 4 mm rostral of the obex on either side; or (3) partial destruction of the dorsal motor (DM) and solitary (SN) nuclei on either side. Additional section of the vagus nerve on the opposite side completely abolished the bradycardiac response. Besides, the GRN bradycardia was also slightly but equally attenuated by making a midline bisection in a length extending from 10 to 4 mm rostral of the obex. Additional section of the vagus nerve on either side abolished completely only the bradycardia following electrical stimulation of the GRN on the same side, while that following electrical stimulation of the G RN on the opposite side remained unaffected. On the other hand, the G R N bradycardia was not affected by simply making a caudal midline bisection in a length extending from 3 or 4 mm rostral to 2 mm caudal of the obex. The results suggest: (1) the ventral part of the G RN is a cardio-inhibitory mechanism independent of the higher center; (2) the efferent pathway descends both ipsilaterally and contralaterally and makes synaptic relay in the areas of DM and/or SN and finally exits via the vagus nerves; (3) the fibers decussate rostral to the level 4 mm rostral of the obex; and (4) both ipsilateral and contralateral descending fibers appeared to exert the same degree of suppressive influence in the heart rate.
222 INTRODUCTION The dorsal motor (DM), solitary (SN), and ambiguus (AN) nuclei are concerned with chemoceptor and/or baroceptor reflex in producing reflex bradycardia via vagus nervest, 14. However, stimulation of the orbital gyri, pyriform lobes, septum, preoptic area, midline thalamic nuclei, the dorsolateral and periventricular regions of the hypothalamus, the reticular formation, central grey and the nucleus of raphe of the midbrain, the caudal pontile reticular nucleus, and the lower part of the medullary gigantocellular reticular nucleus (GRN) could also produce bradycardialA0,19. Neither the physiological functions nor anatomical organizations of these structures have been studied in detail. The nature of the G R N is particularly interesting as it contitutes the most caudal structure which lies closest to the vagal nuclei (DM, SN, and AN) concerned with the basic baroreceptor reflex. Understanding of the G R N will extend understanding of its relation with the rest of the structures in producing vagal bradycardia. The present investigation was performed to localize the areas responsible for bradycardia upon stimulation of the GRN. The descending pathways of this mechanism and its relation with the DM and SN were also studied. METHODS Twenty-six cats of either sex, weighing 2.0-3.5 kg, were anesthetized intraperitoneally by a mixture of chloralose (40 mg/kg) and urethane (400 mg/kg). Tracheas were cannulated for artificial ventilation in case of need. Both sides of vagus nerves were carefully isolated and looped for future use. The right femoral vein was cannulated for fluid supplement and drug administration. The arterial pressure was taken from the right femoral artery through a catheter connected with a Statham P23 AC transducer. The heart rate was computed by a Grass 7P4F tachograph preamplifier triggered by the signal output of arterial pulses from a Grass 7DAF driver amplifier. All recordings were made on a Grass 79D polygraph. The head of the animal was fixed in a D a v i d - K o p f stereotaxic instrument. The cerebellum was partially removed to expose the fourth ventricle. This allowed for more accurate placing of a stimulating electrode into the desired area. The D a v i d - K o p f NEX-100 co-axial electrodes (shaft diameter 0.5 mm, lead diameter 0.2 mm) were used for stimulation. The electrode was mounted on an electrode carrier at an angle of 34°. For localization of the cardio-inhibitory mechanism, systemic exploration of the whole medullary structure rostral to the DM, SN and A N was made. Stimulating current was provided by a Grass $4C square wave stimulator. Stimulating parameters were 30 Hz, 2 msec and 2-4 V. Details of the stimulating technique have been described previously 5. For studying the anatomical connections of the G R N cardio-inhibitory mechanism, the stimulating electrode was usually placed about 8 mm rostral to the obex, 2 mm lateral to the midline, and 4 mm dorsal to the ventral surface of the brain stem. In 3 animals, midcollicular decerebration was performed after the optimal bradycardiac responses were obtained by stimulation of the G R N cardio-inhibitory
223 mechanism. The responses upon the stimulation before and after this procedure were observed and compared. Decerebration interrupted the possible ascending fibers or components connecting with the higher cardio-inhibitory mechanisms. In determining the existence of descending crossed and/or uncrossed fibers from the GRN cardioinhibitory mechanism, paired electrodes, 3.2 mm or 4.0 mm apart, were placed symmetrically into corresponding areas on both sides of the medullary midline. After nearly the same degree of bradycardiac responses were obtained on stimulation of either side, the vagus nerves were cut one after the other. Stimulations were repeated after each section, and the bradycardiac responses were compared. To determine the level of decussation of the crossing fibers, paired electrodes were introduced into the expected location as described above. Midline bisection was done either at the rostral medullary level extending from 2 mm rostral to 4 mm caudal of the stimulated points, or at the caudal level extending from 4 mm rostral to 2 mm caudal of the obex. In addition, in some animals hemisection was done at the level about 4 mm rostral of the obex. The depth of cuttings was about 5 mm. The surgical bisection or hemisection was followed by unilateral and bilateral vagotomies. The bradycardiac responses upon stimulations before and after each procedure were compared. Whether the descending pathways project to the DM and SN of the vagus nerve was determined in 3 animals. The paired electrodes were introduced to the GRN as in the former experiments. After the optimal bradycardiac responses had been obtained by stimulation of both sides, one side of DM and SN was partially ablated by suction with a fine glass pipette (tip diameter about 0.8 mm). Stimulations on each side were repeated. The vagus nerve on the opposite side was then sectioned. Stimulations were again repeated and the bradycardiac responses were compared. After completion of each experiment, the animal was perfused with 1 liter of 0.9 ~o saline solution followed by an equal amount of 10 ~ formalin in saline solution. The brain was removed and further fixed in the same formalin solution for 7-10 days. The stimulated points were identified by frozen sections (20/zm thick) stained with cresyl violet and by Weil's technique. RESULTS Systemic stimulation throughout the medulla oblongata except at its caudal areas which contain the dorsal motor (DM), solitary (SN) and ambiguus (AN) nuclei was carried out with l0 animals. The control heart rate was 195 4- 10 beats/min and arterial pressure 136 4- 9 mm Hg (mean 4- S.E.). The most sensitive area for producing bradycardia was the ventral part of the medullary gigantocellular reticular nucleus (GRN). Stimulating elsewhere out of the area could hardly produce a more than 40 ~ reduction of the heart rate. There were fifty-five points in which stimulation produced a greater reduction of the heart rate than that without any significant rise of the arterial pressure and these are confined in the stippled areas in a composite drawing of the two representative cross-sections (Fig. 1). At level 7-9 mm rostral to the obex, these points were confined in a small area about 1.5-3.5 mm lateral to the
224
ABN'~'~
Fig. 1. Location of the cardio-inhibitory mechanism in GRN of the medulla oblongata in two composite drawings. Upper: 9-7 mm rostral to the obex; lower: 6--4mm rostral to the obex. Division of the scales: 1 mm. Stippled areas indicate stimulation which produced a reduction of the heart rate by more than 40 ~ of the resting value. Abbreviations used: ABN, abducens nucleus; GFN, genu of facial nerve; ION, inferior olivary nucleus; PT, pyramidal tract; SON, superior olivary nucleus; V, spinal trigeminal nucleus and tract.
CAT 0112 Before After Decerebrotlon Deeerebrotion
ARTERIAL
PRESSURE mm H(J
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HEART RATE
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Fig. 2. Bradycardiac responses on left or right GRN stimulation before and after mideollicular decerebration. Note the G R N bradycardia is essentially unchanged before and after the operation.
225 midline a n d 3.5-5.0 m m above the ventral surface. A t level 6.0-4.0 m m rostral to the obex, they were confined in a n area a b o u t 1.0-3.0 m m lateral to the midline a n d 2.0-4.0 m m above the ventral surface. The average reduction o f the heart rate, calculated from these 55 responsive points, was 58.9 ± 3.5 %. The m a x i m a l reduction was 72%. The b r a d y c a r d i a was mostly associated with hypotension. However, it
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30s~c Fig. 3. Effects of unilateral and bilateral vagotomy on the bradycardiac responses elicited by stimulation of two symmetrical points in GRN of the left and right sides in 2 different cats. Points of GRN stimulation, shown in solid circles, were symmetrical. The bradycardiac responses were about equal on either side (control panels). The GRN bradycardia was slightly but equally reduced by unilateral vagotomy either on the left (cat 1224) or right (cat 1222) side. Additional sectioning of the remaining vagus nerve (bilateral vagotomy) completely abolished the GRN bradycardia. Note that in cat 1224 after bilateral vagotomy stimulation of GRN on either side produced a rise instead of lowering of arterial blood pressure, suggesting a concomitant activation of the nearby pressor area.
226 tended to be associated with a rise of arterial pressure when the stimulating voltage was larger than 4 V and/or the stimulated point was localized to the more dorsal and lateral part of the G R N . In 3 animals, interruption of the possible ascending connections of the G R N with the higher centers was done by midcolticular decerebration. The G R N bradycardiac responses before and after the decerebration were essentially similar (Fig. 2). This suggests that the bradycardiac response on stimulation of the G R N resulted mainly from stimulation of the descending efferent fibers directly emerging from the G R N . Stimulation of the descending pathways from the high centers, of course, can not be ruled out. The G R N gives rise to crossed and uncrossed descending fibers mediating bradycardia exclusively via the vagus nerves. As shown in Fig. 3, when the vagi were intact, stimulation of each side of the G R N elicited a similar degree of bradycardia. After either the right (cat 1224) or left (cat 1222) vagus nerve was cut, the bradycardiac responses decreased only slightly but equally upon stimulation of either side. The bradycardia was completely abolished after the remaining vagus nerve was sectioned. A total of 4 animals subject to this group of experiments showed the same results. Midline bisection at the medullary rostral level was done in 2 animals. In one of them (Fig. 4), the bisection extended from 2 m m rostral to 4 m m caudal of the stimulated points (i.e. 10-3.5 m m rostral to the obex). The bisection slightly but equally reduced the bradycardiac responses elicited by stimulation of the G R N on each side. However, further section of the left vagus nerve completely abolished the response upon stimulation of the left G R N , leaving the response of the right G R N
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Fig. 4. Effects of rostral midline bisection (dotted line), left vagotomy, and subsequent right vagotomy on the bradycardiac responses elicited by stimulations of two symmetrical points in GRN of the left and right sides. Note that bisection only slightly but equally reduced the GRN bradycardia of either side. However, after left vagotomy, left GRN stimulation produced no more bradycardia, while right GRN stimulation remained unaffected until subsequent sectioning of the fight vagus nerve.
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Fig. 5. Effects of caudal midline bisection (dotted line), left vagotomy, and subsequent right vagotomy on the bradycardiac responses elicited by stimulations of two symmetrical points in G R N of the left and right sides. This division did not affect the G R N bradycardia of either side. Left vagotomy reduced slightly but equally the G R N bradycardia of either side. Complete abolition was achieved only by subsequent sectioning of the right vagus nerve.
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Fig. 6. Effects of left hemisection and additional right vagotomy on the bradycardiac responses elicited by stimulations of two symmetrical points in G R N of the left and right sides. Hemisection was done on the left side at a level about 4 mm rostral to the obex (dotted line). Hemisection reduced slightly but equally the G R N bradycardia of either side. Additional sectioning of the right vagus nerve completely eliminated the G R N bradycardia. However, with the remains of the left vagus nerve the baroreceptor reflex was still active. A: after left hemisection and right vagotomy, epinephrine 50/~g i.v. elicited a pressor and a reflex bradycardiac response. B: further sectioning of the left vagus nerve abolished the reflex bradycardia.
228 unaffected. Additional right vagotomy subsequently eliminated the bradycardiac response on stimulation of the right GRN. Bisection was done at the caudal level in 2 other animals. As shown in Fig. 5, the bisection extended from 3.5 mm rostral to 2 mm caudal of the obex. The division did not affect the G R N bradycardiac response of either side. Left vagotomy following this bisection slightly reduced the responses upon G R N stimulation on each side. Complete abolition was achieved only after additional section of the right vagus nerve. The findings of these two groups of experiments strongly suggest that crossing fibers of the G R N decussate completely at the rostral medulla, i.e. crossing does not occur more caudal than the level 4 mm rostral of the obex. If this is the case, hemisection at this level by making a transverse cut extending from the midline to either side will completely interrupt all the uncrossing fibers from the G R N of the operated side and the crossed fibers from the opposite side. Two animals were used. As demonstrated in Fig. 6, stimulation of the G R N on either side after left hemisection provoked a smaller bradycardiac response than that before the hemisection. The bradycardiac response on either side was completely eliminated by further section of the right vagus nerve, even though the left vagus nerve and its basic baroreceptor reflex arch mediating reflex bradycardia was still intact. This integrity of the mechanism was demonstrated in Fig. 6A and B. After left hemisection and right vagotomy, epinephrine (50/~g, i.v.) induced an increase of
0721
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Fig. 7. Effects of partial destruction of DM and left vagotomy on the bradycardiac responses elicited by stimulation of two symmetrical points of GRN on the left and right sides. The middle photomicrograph of histological sections stained with cresyl violet shows the center part of the lesion. Note that the rostral part (upper) and caudal part (lower) of the DM remained intact. Also note that the nearby solitary nucleus was inevitably affected. After the lesion, the GRN bradycardia of either side was slightly but equally reduced. Additional left vagotomy completelyeliminated the bradycardia.
229 arterial pressure by about 100 mm Hg and a reflex bradycardia (Fig. 6A). After a further cut of the left vagus nerve, the reflex bradycardia was no longer induced by the same increase of arterial pressure subsequent to the same dose of epinephrine (Fig. 6B). The data from the above 3 groups of experiments thus strongly suggest that crossing fibers of the GRN decussate near the level of their origin. In other words, the most caudally residing basic baroceptor reflex mechanism, i.e. DM, SN and AN, on each side does not transfer the GRN message to the other side. Effect of destruction of the right DM and SN and the left vagotomy on the bradycardiac response upon stimulation of either GRN is shown in Fig. 7. Destruction was only limited at the level of the obex, extending from about 1 mm rostral to 1 mm caudal of the obex. The rostral 1/3 and caudal 1/4 of the nucleus were spared. The lesion, however, inevitably destroyed the SN which is immediately lateral and dorsal to the DM. After the lesion, stimulation of the GRN on either side produced a smaller bradycardiac response in comparison with the control stimulation. Further cutting of the left vagus nerve completely eliminated the bradycardiac response on both sides. This result indicates that the descending projections from the GRN relay in a discrete area around the DM and/or SN. DISCUSSION Findings of the present investigation demonstrated that stimulation of the ventral parts of the GRN produced a bradycardia which could be abolished by bilateral vagotomy, confirming the results of Achari et al. 1. Hypotension was mostly accompanied with the GRN bradycardia and usually abolished by bilateral vagotomy (Figs. 4 and 5). This suggests that the hypotensive response was secondary to bradycardia which reduced the cardiac output, and was not caused by activation of the sympathetic inhibitory mechanism in the medullaa,ls,~9. However, a slight rise of arterial pressure tended to be associated with the bradycardia when the stimulating voltage was high and/or the stimulated point was located to the more dorsal and lateral parts of the GRN. This indicates that pressor mechanisms reside in the vicinity. Actually, pressor vasomotor mechanisms had been localized in the areas dorsal and lateral to the GRNS,~L Thus current spread may be the reason for producing the rise of arterial pressure on the GRN stimulation. The GRN bradycardiac responses were not reflexly triggered by the rise of arterial pressure, as GRN bradycardiac responses occurred mostly concomitant with hypotension or no change in arterial pressure. Increase in arterial pressure was rarely observed. Stimulation of some structures rostral to the GRN provoked bradycardial,~°,~L This raised a question whether activation of the ascending fibers from the GRN or the caudally located vagal nuclei, such as DM, SN and AN, plays a role in the GRN bradycardia. Activation of the higher neural structures may not be the cause of the bradycardia, as separation of the higher centers from the GRN by midcollicular decerebration did not affect the GRN bradycardia (Fig. 2). This, however, does not rule out the possibility that GRN contains ascending cardio-inhibitory fibers to higher neural structures. Further investigation has to be done in answering this question. The present experiment shows that the GRN cardio-inhibitory mechanism gives,
230 rise to both uncrossed and crossed descending fibers mediating bradycardiac response via both sides of the vagus nerves. In addition, the results also reveal that both uncrossed and crossed projections offer the same degree of suppressive influence in the heart rate. These were supported by the evidence that the bradycardiac response on the G R N stimulation of either side was slightly but equally reduced by: (1) sectioning either side of the vagus nerve (Fig. 3); (2) hemisection on either side of the medulla (Fig. 6); or (3) partial destruction of the DM and SN on either side (Fig. 7). Further, complete abolition of the bradycardiac response was achieved when the vagus nerve opposite to the above surgical side was cut. In addition, similar attenuation of the G R N bradycardia occurred when a rostral midline bisection was done, while further section of the vagus nerve on either side only abolished the bradycardiac response upon stimulation of the G R N on the same side, but the response upon stimulation of the other side remained not eliminated until section of the rest of the vagus nerve (Fig. 4), supporting also the abovementioned suggestion. The second important finding is that the G R N crossing fibers decussate completely rostral to the level 4 mm rostral of the obex. This was demonstrated in Figs. 4, 5 and 6. In Fig. 4, after a rostral midline bisection the bradycardiac response on stimulating one side of the G R N was abolished by sectioning the same side of the vagus nerve. On the other hand, the bradycardiac response, on stimulating either side of the GRN, was not affected simply by a caudal midline bisection (Fig. 5). In Fig. 6, after left hemisection (at level of 4 mm rostrat of the obex), stimulation of the left G R N elicited a similar degree of bradycardiac response as stimulation of the right G R N did. This indicates that crossing fibers from the left G R N were not interrupted by the hemisection. However, after the left hemisection, crossed fibers from the right G R N (and also the uncrossed fibers from the left GRN) were completely severed. As a result, after additional section of the right vagus nerve, interrupting the uncrossed fibers from the right GRN, no more bradycardia was observed by stimulation of the right GRN. It has been claimed that the cardio-inhibitory mechanism of the SN or AN projects to the ipsilateral and contralateral vagus nerves because the bradycardia induced by stimulation of these nuclei was not completely abolished by ipsilaterat vagotomy 4,9. Fibers were presumed passing across the caudally residing commissural nuclei of Cajal. This is entirely different with the crossing pathway of the GRN. It is still not known whether the commissural nuclei or the G R N relays the bilateral projections of the baroreceptive reflex bradycardia 11. The third important finding is that the G R N descending projections make synaptic relay in the areas of the DM and SN. The evidence is based on the observation that partial destruction of the DM and SN on either side reduced the bradycardia on stimulation of the G R N on either side. Complete abolition of the bradycardia was observed after additional section of the vagus nerve on the other side (Fig. 7). The precise location of the cardioqnhibitory preganglionic neurons is a matter of dispute as some investigators suggested that cardio-inhibitory preganglionic fibers of the vagus nerve originate from the DM7,8,13,15, while others suggested the AN 2,5,9,1~.
231
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.__.__/
Fig. 8. A schematic diagram illustrating the possible GRN crossing and uncrossing descending fibers in relation with the dorsal motor nucleus of the vagus (DM) and/or solitary (SN) nuclei and the preganglionic cardio-inhibitory fibers (PCF). Most recently, Todo et al. 17, using the horseradish peroxidase technique, observed that the vagal preganglionic fibers to the sinoatrial and atrioventricular node regions originate in the areas of the D M and the caudal medial solitary nucleus. In the present experiment, the lesion was made at the caudal parts of the D M and SN based on the findings of Gets and Sirnes s as well as T o d o et al.lL Thus the results of the present experiment suggest an anatomical connection between the G R N and the cardio-inhibitory mechanism of the D M and SN. Whether the D M or SN alone, or both, give rise to the preganglionic vagal fibers which receive the G R N projections, however, could not be determined, because the D M and SN are so closely located that destruction of either one will inevitably involve the other. On the basis of the present findings and results in the literature, a schematic diagram (Fig. 8) is drawn to illustrate the possible G R N bradycardiac pathways in relation to the D M and SN. The ventral part of the G R N gives rise to both crossed and uncrossed descending fibers projecting to the D M and/or SN which in turn sends efferent preganglionic cardio-inhibitory fibers to the heart s,~7. ACKNOWLEDGEMENT This project was supported in part by grants from the National Science Council, Republic of China and the J. A r o n Foundation, U.S.A.
REFERENCES 1 Achari, N. K., Downman, C. B. B. and Weber, W. B., A cardioinhibitory pathway in the brain stem of the cat, J. Physiol. (Lond.), 197 (1968) 35P. 2 Borison, H. L. and Domjan, D., Persistence of the cardio-inhibitory response to brain stem ischemia after destruction of the area postrema and the dorsal vagal nuclei, J. Physiol. (Lond.), 211 (1970) 263-277. 3 Calaresu, F. R. and Thomas, M. R., The function of the paramedian reticular nucleus in the control of heart rate in the cat, J. Physiol. (Lond.), 216 (1971) 143-158.
232 4 Calaresu, F. R. and Pearce, J. W., Effects on heart rate of electrical stimulation of medullary vagal structures in the cat, J. Physiol. (Lond.), 176 (1965) 241-251. 5 Chai, C.Y. andWang, S. C.,Localizationofcentralcardiovascularcontrolmechanisminthelower brain stem of the cat, Amer. J. Physiol., 202 (1962) 25-30. 6 Chert, H. 1. and Chai, C. Y., Integration of the cardiovagal mechanism in the medulla oblongata of the cat, Amer. J. PhysioL, 231 (1976) 454-461. 7 Cohen, D. H. and Schenall, A. M., Medullary cells of origin of vagal cardioinhibitory fibers in the pigeon. II. Electrical stimulation of the dorsal motor nucleus, J. comp. Neurol., 140 (1970) 321-342. 8 Getz, B. and Sirnes, T., The localization with the dorsal motor vagal nucleus, J. comp. Neurol., 90 (1949) 95-110. 9 Gunn, C. G., Sevelius, G., Puiggari, M. J. and Myer, F. K., Vagal cardiomotor mechanisms in the hindbrain of the dog and cat, Amer. J. Physiol., 214 (1968) 258-262. 10 Korteweg, G. C. J., Boeles, J. Th. F. and Cate, J. Ten., Influence of stimulation of some subcortical areas on electrocardiogram, J. Neurophysiol., 20 (1957) 100-107. 11 Kardon, M. B., Peterson,D. F. and Bishop,V. S., Reflex bradycardia due to aortic nerve stimulation in the rabbit, Amer. J. Physiol., 225 (1973) 7-11. 12 Kuo, J. S., Chai, C. Y., Lee, T. M., Liu, C. N. and Lim, R. K. S., Localization of central cardiovascular control mechanism in the brain stem of the monkey, Exp. NeuroL, 29 (1970) 131-141. 13 Mitchell, G. A. G. and Warwick, R., The dorsal motor nucleus, Acta anat. (Basel), 25 (1955) 371395. 14 Lee, T. M., Kuo, J. S. and Chai, C. Y., Central integrating mechanism of the Bezold-Jarisch and baroceptor reflexes, Amer. J. Physiol., 222 (1972) 713-720. 15 Schwaber, J. S. and Cohen, D. H., Localization and identification of vagal cardioinhibitoryneurons in the pigeon. In Society for Neuroscience, Sixth Annual Meeting, Vol. 11, Part 1, 1976, 79 pp. 16 Thomas, M. R. and Calaresu, F. R., Localization and function of medullary sites mediating vagal bradycardia, Amer. J. PhysioL, 226 (1974) 1344-1349. 17 Todo, K., Yamamoto, T., Satomi, H., lse, H., Takatama, H. and Takahashi, K., Origins of vagal preganglionic fibers to the sino-atrial and atrioventricularnode regions in the cat heart as studied by the horseradish peroxidase method, Brain Research, 130 (1977) 545-550. 18 Wang, S. C., Bulbar regulatory mechanism. In Price, H. L. and Cohen, J. (Eds.), Effects of Anesthetics on the Circulation, Thomas, Springfield, 111., 1964, pp. 16--31. 19 Wang, S. C. and Ranson, S. W., The role of the hypothalamus and preoptic region in the regulation of heart rate, Amer. J. Physiol., 132 (1941) 5-8.
221
© Elsevier/North-HollandBiomedicalPress
CARDIO-INHIBITORY MECHANISM IN THE GIGANTOCELLULAR RETICULAR NUCLEUS OF THE MEDULLA OBLONGATA
J. S. KUO, Y. HWA and C. Y. CHAI Department of Physiology and Biophysics, National Defense Medical Center, and Kohlberg Memorial Medical ResearchLaboratory, Veterans GeneralHospital, Taipei (Taiwan)
(Accepted April 5th, 1979) Key words:
cardio-inhibitory mechanism - - gigantocellular reticular nucleus - bradycardia - - vagus nerve
SUMMARY A cardio-inhibitory mechanism was localized in the ventral part of the medullary gigantocellular reticular nucleus (GRN) in chloralose-urethane anesthetized cats. Stimulation of this mechanism produced an average 58.9 ~ reduction of the heart rate (calculated from 55 responsive points having more than 4 0 ~ reduction) associated mostly with hypotension, or no change or occasionally a slight increase of the arterial blood pressure. Midcollicular decerebration did not affect this bradycardia. The bradycardia following G R N stimulation of either side by a pair of symmetrically placed electrodes was reduced slightly but equally by: (1) sectioning either side of the vagus nerve; (2) hemisection at a level 4 mm rostral of the obex on either side; or (3) partial destruction of the dorsal motor (DM) and solitary (SN) nuclei on either side. Additional section of the vagus nerve on the opposite side completely abolished the bradycardiac response. Besides, the GRN bradycardia was also slightly but equally attenuated by making a midline bisection in a length extending from 10 to 4 mm rostral of the obex. Additional section of the vagus nerve on either side abolished completely only the bradycardia following electrical stimulation of the GRN on the same side, while that following electrical stimulation of the G RN on the opposite side remained unaffected. On the other hand, the G R N bradycardia was not affected by simply making a caudal midline bisection in a length extending from 3 or 4 mm rostral to 2 mm caudal of the obex. The results suggest: (1) the ventral part of the G RN is a cardio-inhibitory mechanism independent of the higher center; (2) the efferent pathway descends both ipsilaterally and contralaterally and makes synaptic relay in the areas of DM and/or SN and finally exits via the vagus nerves; (3) the fibers decussate rostral to the level 4 mm rostral of the obex; and (4) both ipsilateral and contralateral descending fibers appeared to exert the same degree of suppressive influence in the heart rate.
222 INTRODUCTION The dorsal motor (DM), solitary (SN), and ambiguus (AN) nuclei are concerned with chemoceptor and/or baroceptor reflex in producing reflex bradycardia via vagus nervest, 14. However, stimulation of the orbital gyri, pyriform lobes, septum, preoptic area, midline thalamic nuclei, the dorsolateral and periventricular regions of the hypothalamus, the reticular formation, central grey and the nucleus of raphe of the midbrain, the caudal pontile reticular nucleus, and the lower part of the medullary gigantocellular reticular nucleus (GRN) could also produce bradycardialA0,19. Neither the physiological functions nor anatomical organizations of these structures have been studied in detail. The nature of the G R N is particularly interesting as it contitutes the most caudal structure which lies closest to the vagal nuclei (DM, SN, and AN) concerned with the basic baroreceptor reflex. Understanding of the G R N will extend understanding of its relation with the rest of the structures in producing vagal bradycardia. The present investigation was performed to localize the areas responsible for bradycardia upon stimulation of the GRN. The descending pathways of this mechanism and its relation with the DM and SN were also studied. METHODS Twenty-six cats of either sex, weighing 2.0-3.5 kg, were anesthetized intraperitoneally by a mixture of chloralose (40 mg/kg) and urethane (400 mg/kg). Tracheas were cannulated for artificial ventilation in case of need. Both sides of vagus nerves were carefully isolated and looped for future use. The right femoral vein was cannulated for fluid supplement and drug administration. The arterial pressure was taken from the right femoral artery through a catheter connected with a Statham P23 AC transducer. The heart rate was computed by a Grass 7P4F tachograph preamplifier triggered by the signal output of arterial pulses from a Grass 7DAF driver amplifier. All recordings were made on a Grass 79D polygraph. The head of the animal was fixed in a D a v i d - K o p f stereotaxic instrument. The cerebellum was partially removed to expose the fourth ventricle. This allowed for more accurate placing of a stimulating electrode into the desired area. The D a v i d - K o p f NEX-100 co-axial electrodes (shaft diameter 0.5 mm, lead diameter 0.2 mm) were used for stimulation. The electrode was mounted on an electrode carrier at an angle of 34°. For localization of the cardio-inhibitory mechanism, systemic exploration of the whole medullary structure rostral to the DM, SN and A N was made. Stimulating current was provided by a Grass $4C square wave stimulator. Stimulating parameters were 30 Hz, 2 msec and 2-4 V. Details of the stimulating technique have been described previously 5. For studying the anatomical connections of the G R N cardio-inhibitory mechanism, the stimulating electrode was usually placed about 8 mm rostral to the obex, 2 mm lateral to the midline, and 4 mm dorsal to the ventral surface of the brain stem. In 3 animals, midcollicular decerebration was performed after the optimal bradycardiac responses were obtained by stimulation of the G R N cardio-inhibitory
223 mechanism. The responses upon the stimulation before and after this procedure were observed and compared. Decerebration interrupted the possible ascending fibers or components connecting with the higher cardio-inhibitory mechanisms. In determining the existence of descending crossed and/or uncrossed fibers from the GRN cardioinhibitory mechanism, paired electrodes, 3.2 mm or 4.0 mm apart, were placed symmetrically into corresponding areas on both sides of the medullary midline. After nearly the same degree of bradycardiac responses were obtained on stimulation of either side, the vagus nerves were cut one after the other. Stimulations were repeated after each section, and the bradycardiac responses were compared. To determine the level of decussation of the crossing fibers, paired electrodes were introduced into the expected location as described above. Midline bisection was done either at the rostral medullary level extending from 2 mm rostral to 4 mm caudal of the stimulated points, or at the caudal level extending from 4 mm rostral to 2 mm caudal of the obex. In addition, in some animals hemisection was done at the level about 4 mm rostral of the obex. The depth of cuttings was about 5 mm. The surgical bisection or hemisection was followed by unilateral and bilateral vagotomies. The bradycardiac responses upon stimulations before and after each procedure were compared. Whether the descending pathways project to the DM and SN of the vagus nerve was determined in 3 animals. The paired electrodes were introduced to the GRN as in the former experiments. After the optimal bradycardiac responses had been obtained by stimulation of both sides, one side of DM and SN was partially ablated by suction with a fine glass pipette (tip diameter about 0.8 mm). Stimulations on each side were repeated. The vagus nerve on the opposite side was then sectioned. Stimulations were again repeated and the bradycardiac responses were compared. After completion of each experiment, the animal was perfused with 1 liter of 0.9 ~o saline solution followed by an equal amount of 10 ~ formalin in saline solution. The brain was removed and further fixed in the same formalin solution for 7-10 days. The stimulated points were identified by frozen sections (20/zm thick) stained with cresyl violet and by Weil's technique. RESULTS Systemic stimulation throughout the medulla oblongata except at its caudal areas which contain the dorsal motor (DM), solitary (SN) and ambiguus (AN) nuclei was carried out with l0 animals. The control heart rate was 195 4- 10 beats/min and arterial pressure 136 4- 9 mm Hg (mean 4- S.E.). The most sensitive area for producing bradycardia was the ventral part of the medullary gigantocellular reticular nucleus (GRN). Stimulating elsewhere out of the area could hardly produce a more than 40 ~ reduction of the heart rate. There were fifty-five points in which stimulation produced a greater reduction of the heart rate than that without any significant rise of the arterial pressure and these are confined in the stippled areas in a composite drawing of the two representative cross-sections (Fig. 1). At level 7-9 mm rostral to the obex, these points were confined in a small area about 1.5-3.5 mm lateral to the
224
ABN'~'~
Fig. 1. Location of the cardio-inhibitory mechanism in GRN of the medulla oblongata in two composite drawings. Upper: 9-7 mm rostral to the obex; lower: 6--4mm rostral to the obex. Division of the scales: 1 mm. Stippled areas indicate stimulation which produced a reduction of the heart rate by more than 40 ~ of the resting value. Abbreviations used: ABN, abducens nucleus; GFN, genu of facial nerve; ION, inferior olivary nucleus; PT, pyramidal tract; SON, superior olivary nucleus; V, spinal trigeminal nucleus and tract.
CAT 0112 Before After Decerebrotlon Deeerebrotion
ARTERIAL
PRESSURE mm H(J
200, I O0 tlllml~ 0
HEART RATE
~
Left Side
[ f ii r /mlm................ t
240[ beats/mln
Slim
'
801
ARTERIAL
PRESSURE m m Hg
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~
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.
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.
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.
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Fig. 2. Bradycardiac responses on left or right GRN stimulation before and after mideollicular decerebration. Note the G R N bradycardia is essentially unchanged before and after the operation.
225 midline a n d 3.5-5.0 m m above the ventral surface. A t level 6.0-4.0 m m rostral to the obex, they were confined in a n area a b o u t 1.0-3.0 m m lateral to the midline a n d 2.0-4.0 m m above the ventral surface. The average reduction o f the heart rate, calculated from these 55 responsive points, was 58.9 ± 3.5 %. The m a x i m a l reduction was 72%. The b r a d y c a r d i a was mostly associated with hypotension. However, it
CAT 1224
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30s~c Fig. 3. Effects of unilateral and bilateral vagotomy on the bradycardiac responses elicited by stimulation of two symmetrical points in GRN of the left and right sides in 2 different cats. Points of GRN stimulation, shown in solid circles, were symmetrical. The bradycardiac responses were about equal on either side (control panels). The GRN bradycardia was slightly but equally reduced by unilateral vagotomy either on the left (cat 1224) or right (cat 1222) side. Additional sectioning of the remaining vagus nerve (bilateral vagotomy) completely abolished the GRN bradycardia. Note that in cat 1224 after bilateral vagotomy stimulation of GRN on either side produced a rise instead of lowering of arterial blood pressure, suggesting a concomitant activation of the nearby pressor area.
226 tended to be associated with a rise of arterial pressure when the stimulating voltage was larger than 4 V and/or the stimulated point was localized to the more dorsal and lateral part of the G R N . In 3 animals, interruption of the possible ascending connections of the G R N with the higher centers was done by midcolticular decerebration. The G R N bradycardiac responses before and after the decerebration were essentially similar (Fig. 2). This suggests that the bradycardiac response on stimulation of the G R N resulted mainly from stimulation of the descending efferent fibers directly emerging from the G R N . Stimulation of the descending pathways from the high centers, of course, can not be ruled out. The G R N gives rise to crossed and uncrossed descending fibers mediating bradycardia exclusively via the vagus nerves. As shown in Fig. 3, when the vagi were intact, stimulation of each side of the G R N elicited a similar degree of bradycardia. After either the right (cat 1224) or left (cat 1222) vagus nerve was cut, the bradycardiac responses decreased only slightly but equally upon stimulation of either side. The bradycardia was completely abolished after the remaining vagus nerve was sectioned. A total of 4 animals subject to this group of experiments showed the same results. Midline bisection at the medullary rostral level was done in 2 animals. In one of them (Fig. 4), the bisection extended from 2 m m rostral to 4 m m caudal of the stimulated points (i.e. 10-3.5 m m rostral to the obex). The bisection slightly but equally reduced the bradycardiac responses elicited by stimulation of the G R N on each side. However, further section of the left vagus nerve completely abolished the response upon stimulation of the left G R N , leaving the response of the right G R N
CAT 0 6 1 0
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Fig. 4. Effects of rostral midline bisection (dotted line), left vagotomy, and subsequent right vagotomy on the bradycardiac responses elicited by stimulations of two symmetrical points in GRN of the left and right sides. Note that bisection only slightly but equally reduced the GRN bradycardia of either side. However, after left vagotomy, left GRN stimulation produced no more bradycardia, while right GRN stimulation remained unaffected until subsequent sectioning of the fight vagus nerve.
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Fig. 5. Effects of caudal midline bisection (dotted line), left vagotomy, and subsequent right vagotomy on the bradycardiac responses elicited by stimulations of two symmetrical points in G R N of the left and right sides. This division did not affect the G R N bradycardia of either side. Left vagotomy reduced slightly but equally the G R N bradycardia of either side. Complete abolition was achieved only by subsequent sectioning of the right vagus nerve.
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Fig. 6. Effects of left hemisection and additional right vagotomy on the bradycardiac responses elicited by stimulations of two symmetrical points in G R N of the left and right sides. Hemisection was done on the left side at a level about 4 mm rostral to the obex (dotted line). Hemisection reduced slightly but equally the G R N bradycardia of either side. Additional sectioning of the right vagus nerve completely eliminated the G R N bradycardia. However, with the remains of the left vagus nerve the baroreceptor reflex was still active. A: after left hemisection and right vagotomy, epinephrine 50/~g i.v. elicited a pressor and a reflex bradycardiac response. B: further sectioning of the left vagus nerve abolished the reflex bradycardia.
228 unaffected. Additional right vagotomy subsequently eliminated the bradycardiac response on stimulation of the right GRN. Bisection was done at the caudal level in 2 other animals. As shown in Fig. 5, the bisection extended from 3.5 mm rostral to 2 mm caudal of the obex. The division did not affect the G R N bradycardiac response of either side. Left vagotomy following this bisection slightly reduced the responses upon G R N stimulation on each side. Complete abolition was achieved only after additional section of the right vagus nerve. The findings of these two groups of experiments strongly suggest that crossing fibers of the G R N decussate completely at the rostral medulla, i.e. crossing does not occur more caudal than the level 4 mm rostral of the obex. If this is the case, hemisection at this level by making a transverse cut extending from the midline to either side will completely interrupt all the uncrossing fibers from the G R N of the operated side and the crossed fibers from the opposite side. Two animals were used. As demonstrated in Fig. 6, stimulation of the G R N on either side after left hemisection provoked a smaller bradycardiac response than that before the hemisection. The bradycardiac response on either side was completely eliminated by further section of the right vagus nerve, even though the left vagus nerve and its basic baroreceptor reflex arch mediating reflex bradycardia was still intact. This integrity of the mechanism was demonstrated in Fig. 6A and B. After left hemisection and right vagotomy, epinephrine (50/~g, i.v.) induced an increase of
0721
CAT
ARTERIAL PRESSURE
mm Hg
Control
2000 ~ J0 ~
240[-
~
• /
HEART RATE beats/rain
ARTERIAL PRESSURE
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After Rt. DMN After Lt. Destruction Vagotomy
I 60 80
.
.
.
Im~l,m~ ~/--
.
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.....
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,
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RATE beots/min
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~,,~. , f
.~......
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I 6 8
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Fig. 7. Effects of partial destruction of DM and left vagotomy on the bradycardiac responses elicited by stimulation of two symmetrical points of GRN on the left and right sides. The middle photomicrograph of histological sections stained with cresyl violet shows the center part of the lesion. Note that the rostral part (upper) and caudal part (lower) of the DM remained intact. Also note that the nearby solitary nucleus was inevitably affected. After the lesion, the GRN bradycardia of either side was slightly but equally reduced. Additional left vagotomy completelyeliminated the bradycardia.
229 arterial pressure by about 100 mm Hg and a reflex bradycardia (Fig. 6A). After a further cut of the left vagus nerve, the reflex bradycardia was no longer induced by the same increase of arterial pressure subsequent to the same dose of epinephrine (Fig. 6B). The data from the above 3 groups of experiments thus strongly suggest that crossing fibers of the GRN decussate near the level of their origin. In other words, the most caudally residing basic baroceptor reflex mechanism, i.e. DM, SN and AN, on each side does not transfer the GRN message to the other side. Effect of destruction of the right DM and SN and the left vagotomy on the bradycardiac response upon stimulation of either GRN is shown in Fig. 7. Destruction was only limited at the level of the obex, extending from about 1 mm rostral to 1 mm caudal of the obex. The rostral 1/3 and caudal 1/4 of the nucleus were spared. The lesion, however, inevitably destroyed the SN which is immediately lateral and dorsal to the DM. After the lesion, stimulation of the GRN on either side produced a smaller bradycardiac response in comparison with the control stimulation. Further cutting of the left vagus nerve completely eliminated the bradycardiac response on both sides. This result indicates that the descending projections from the GRN relay in a discrete area around the DM and/or SN. DISCUSSION Findings of the present investigation demonstrated that stimulation of the ventral parts of the GRN produced a bradycardia which could be abolished by bilateral vagotomy, confirming the results of Achari et al. 1. Hypotension was mostly accompanied with the GRN bradycardia and usually abolished by bilateral vagotomy (Figs. 4 and 5). This suggests that the hypotensive response was secondary to bradycardia which reduced the cardiac output, and was not caused by activation of the sympathetic inhibitory mechanism in the medullaa,ls,~9. However, a slight rise of arterial pressure tended to be associated with the bradycardia when the stimulating voltage was high and/or the stimulated point was located to the more dorsal and lateral parts of the GRN. This indicates that pressor mechanisms reside in the vicinity. Actually, pressor vasomotor mechanisms had been localized in the areas dorsal and lateral to the GRNS,~L Thus current spread may be the reason for producing the rise of arterial pressure on the GRN stimulation. The GRN bradycardiac responses were not reflexly triggered by the rise of arterial pressure, as GRN bradycardiac responses occurred mostly concomitant with hypotension or no change in arterial pressure. Increase in arterial pressure was rarely observed. Stimulation of some structures rostral to the GRN provoked bradycardial,~°,~L This raised a question whether activation of the ascending fibers from the GRN or the caudally located vagal nuclei, such as DM, SN and AN, plays a role in the GRN bradycardia. Activation of the higher neural structures may not be the cause of the bradycardia, as separation of the higher centers from the GRN by midcollicular decerebration did not affect the GRN bradycardia (Fig. 2). This, however, does not rule out the possibility that GRN contains ascending cardio-inhibitory fibers to higher neural structures. Further investigation has to be done in answering this question. The present experiment shows that the GRN cardio-inhibitory mechanism gives,
230 rise to both uncrossed and crossed descending fibers mediating bradycardiac response via both sides of the vagus nerves. In addition, the results also reveal that both uncrossed and crossed projections offer the same degree of suppressive influence in the heart rate. These were supported by the evidence that the bradycardiac response on the G R N stimulation of either side was slightly but equally reduced by: (1) sectioning either side of the vagus nerve (Fig. 3); (2) hemisection on either side of the medulla (Fig. 6); or (3) partial destruction of the DM and SN on either side (Fig. 7). Further, complete abolition of the bradycardiac response was achieved when the vagus nerve opposite to the above surgical side was cut. In addition, similar attenuation of the G R N bradycardia occurred when a rostral midline bisection was done, while further section of the vagus nerve on either side only abolished the bradycardiac response upon stimulation of the G R N on the same side, but the response upon stimulation of the other side remained not eliminated until section of the rest of the vagus nerve (Fig. 4), supporting also the abovementioned suggestion. The second important finding is that the G R N crossing fibers decussate completely rostral to the level 4 mm rostral of the obex. This was demonstrated in Figs. 4, 5 and 6. In Fig. 4, after a rostral midline bisection the bradycardiac response on stimulating one side of the G R N was abolished by sectioning the same side of the vagus nerve. On the other hand, the bradycardiac response, on stimulating either side of the GRN, was not affected simply by a caudal midline bisection (Fig. 5). In Fig. 6, after left hemisection (at level of 4 mm rostrat of the obex), stimulation of the left G R N elicited a similar degree of bradycardiac response as stimulation of the right G R N did. This indicates that crossing fibers from the left G R N were not interrupted by the hemisection. However, after the left hemisection, crossed fibers from the right G R N (and also the uncrossed fibers from the left GRN) were completely severed. As a result, after additional section of the right vagus nerve, interrupting the uncrossed fibers from the right GRN, no more bradycardia was observed by stimulation of the right GRN. It has been claimed that the cardio-inhibitory mechanism of the SN or AN projects to the ipsilateral and contralateral vagus nerves because the bradycardia induced by stimulation of these nuclei was not completely abolished by ipsilaterat vagotomy 4,9. Fibers were presumed passing across the caudally residing commissural nuclei of Cajal. This is entirely different with the crossing pathway of the GRN. It is still not known whether the commissural nuclei or the G R N relays the bilateral projections of the baroreceptive reflex bradycardia 11. The third important finding is that the G R N descending projections make synaptic relay in the areas of the DM and SN. The evidence is based on the observation that partial destruction of the DM and SN on either side reduced the bradycardia on stimulation of the G R N on either side. Complete abolition of the bradycardia was observed after additional section of the vagus nerve on the other side (Fig. 7). The precise location of the cardioqnhibitory preganglionic neurons is a matter of dispute as some investigators suggested that cardio-inhibitory preganglionic fibers of the vagus nerve originate from the DM7,8,13,15, while others suggested the AN 2,5,9,1~.
231
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Fig. 8. A schematic diagram illustrating the possible GRN crossing and uncrossing descending fibers in relation with the dorsal motor nucleus of the vagus (DM) and/or solitary (SN) nuclei and the preganglionic cardio-inhibitory fibers (PCF). Most recently, Todo et al. 17, using the horseradish peroxidase technique, observed that the vagal preganglionic fibers to the sinoatrial and atrioventricular node regions originate in the areas of the D M and the caudal medial solitary nucleus. In the present experiment, the lesion was made at the caudal parts of the D M and SN based on the findings of Gets and Sirnes s as well as T o d o et al.lL Thus the results of the present experiment suggest an anatomical connection between the G R N and the cardio-inhibitory mechanism of the D M and SN. Whether the D M or SN alone, or both, give rise to the preganglionic vagal fibers which receive the G R N projections, however, could not be determined, because the D M and SN are so closely located that destruction of either one will inevitably involve the other. On the basis of the present findings and results in the literature, a schematic diagram (Fig. 8) is drawn to illustrate the possible G R N bradycardiac pathways in relation to the D M and SN. The ventral part of the G R N gives rise to both crossed and uncrossed descending fibers projecting to the D M and/or SN which in turn sends efferent preganglionic cardio-inhibitory fibers to the heart s,~7. ACKNOWLEDGEMENT This project was supported in part by grants from the National Science Council, Republic of China and the J. A r o n Foundation, U.S.A.
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