Brain Research, 568 (1991) 165-172 (~ 1991 Elsevier Science Publishers B.V. All fights reserved. 0006-8993/91/$03.50
165
RES 17293
Localization of respiratory bulbospinal and propriobulbar neurons in the region of the nucleus ambiguus of the rat P.A. Nfifiez-Abades 1, R.
P~isaro I a n d A . L . Bianchi 2
1Laboratory of Neuroscience, Department of Animal Physiology and Biology, University of Sevilla, SeviUa (Spain) and ZD#partement de Physiologie et Neurophysiologie, C.N.R.S. URA 205, Facuh# des Sciences et Techniques, Marseille (France)
(Accepted 13 August 1991) Key words: Bttzinger complex; Diamidino yellow; Esophagus; Expiratory neuron; Fast blue; Fluorescent marker; Fluorogold; Nucleus ambiguus; Phrenic nucleus; Rat; Swallowing muscle; Vagal motoneuron; Ventral respiratory group
The location of neurons within the ventral respiratory group (VRG) of rat was mapped following injections of 3 different fluorochrome tracers into different rues known to receive projections from VRG neurons. Injection sites included muscles innervated by the vagus (X) and glossopharyngeal (IX) nerves, and the sites of expiratory activity in the caudal medulla and of inspiratory activity in the spinal cord at the C4 level. Labeling of vagal motoneurous resulting from fluorochrome injections into muscles innervated by X and IX nerves was always ipsilateral to the site of injection. Both propfiobulbar and bulbospinal neurons had primarily ipsilaterai projections. No double-labeled cell bodies were observed. The cell bodies of the 3 types of neurons, propfiobulbar, bulbospinal and vagai/glossopharyngeai,were unevenly distributed along the rostrocaudal axis of the VRG, suggesting a complex mosaic of neurons which regulate respiratory-related functions such as swallowing and vocahzation.
INTRODUCTION The nucleus ambiguus (NA) of the rat consists of a longitudinal cell column of large multipolar neurons lying ventral to the parvocellular reticular nucleus and dorsal to the lateral reticular nucleus. It extends from the facial nucleus as far caudally as the pyramidal decussation 3. Although classically described as being composed of large motoneurons (Mns) which innervate the laryngeal and pharyngeal muscles 17, further studies have demonstrated the existence of a viscerotopic organization TM 12.24. The rostral part contains esophageal Mns (compact formation), the intermediate part contains laryngeal and pharyngeal Mns (semicompact formation), and the caudal part contains laryngeal Mns (loose formation). Ventral and dorsal to this motoneuronal pool, but external to it, lies an aggregation of preganglionic neurons innervating the heart and other supradiaphragmatic structures 2. Morphological and electrophysiological studies have demonstrated the existence of a ventral respiratory group (VRG) of neurons associated with the NA. The V R G contains bulbospinal neurons which provide the premotor control to phrenic, intercostal and abdominal motoneurons 4'11'22. It also contains propriobulbar neurons
interconnected to neurons in other brainstem respiratory groups 5'2°. In the rat V R O , 3 different subgroups of respiratory neurons have been found: (1) the BOtzinger complex (BC), a cluster of neurons in the most rostral part of the V R G located ventromedial to the rostral part of NA, consists primarily of expiratory neurons, (2) the intermediate part of the V R G , rostral to the obex (rVRG), contains mostly inspiratory neurons; and (3) caudal to the obex the caudal division of V R G (cVRG) contains mostly expiratory neurons 6'a'26'29. The anatomical organization of the NA likely provides the structural basis for its functional role in controlling breathing and such other related functions as sneezing, coughing, swallowing, gagging, phonation, and vomiting. We therefore examined the location, morphology, and orientation of the dendritic trees of the 3 different populations of neurons in the N A and the surrounding reticular region which constitute the V R G , i.e. bulbospinal neurons projeering to phrenic Mns, propriobulbar interneurons projecting to expiratory neurons of the cVRG, and vagal and glossopharyngeal neurons projecting to the pharyngeal and laryngeal muscles, and to the esophageal muscles. We investigated both sides of the medulla, allowing us to examine the bilateral organization of the neuronal networks involved in these respiratory-related
Correspondence: R. P~saxo, Laboratorio de Neurociencia, Departemento de Fisiologia y Biologta Animal, Facultad de Biologta, Avda. R¢ina Mercedes 6, 41012-Sevilla, Spain.
166 functions but also to d e t e r m i n e the r e l a t i v e strengths of the ipsilateral and c o n t r a l a t e r a l p r o j e c t i o n s , a topic not e x p l o r e d in p r e v i o u s studies 6'2a. Lastly, t h e s e experim e n t s a l l o w e d us to test for the existence o f possible axonal collateral projections.
MATERIALS AND METHODS Expenments were carried out on 15 albino Wistar rats weighing 250-400 g. The animals were anesthetized with ketamine (50 mg/kg Lp.). In addition, atropine sulfate (0,5 mg/kg i.m.) was admimstered to reduce mucus secretions. Subsequent maintenance doses of ketamine were administered i.m. when needed Rectal temperature was mmntalned at 37-38°C.
Surgical procedures To label the motoneurons of the NA on both sides of medulla, the following muscles and nerves were injected bilaterally: the caudal and medial pharyngeal constrictor muscles, innervated by the pharyngeal branch of the vagus nerve; the stylopharyngeal muscle innervated by the glossopharyngeal nerve (IX); the cricothyroid muscle innervated by the superior laryngeal nerve, and cervical vagus nerve (X) which innervates through the recurrent laryngeal nerve, the posterior cricoarytenoid, lateral cricoarytenoid and thyroarytenoid muscles of the larynx, and the esophagus. Injections of the tracers were made through micropipettes attached to a pressure injection apparatus (Picospritzer) and under direct visual observation using a Zeiss dissecting microscope. The microplpettes (100-200 #m tip diameter) were filled with a 2% fluorogold solution made m sahne (FG, Fluorochrome Inc., Englewood Co.) 27 For injections into muscles, several injections were made until the muscles lost their color. In nerves, injections were made at low pressures after the micropipette tip had been inserted through the perineurium into the nerves. Following injections, the animals were allowed to recover. AmpieiUin (10,000 IU i.m.) was administered Three days later, the animals were anesthetized again as previously described. We monitored central respiratory activity, by means of two recording electrodes (insulated silver wire, 200/tm diameter) which were inserted into the diaphragm from the abdominal side. The signal was amplified and displayed on an oscilloscope. In order to localize bulbospinal neurons in the VRG projecting to the phrenic nucleus, we recorded activity of the phremc motoneurons via a micropipette which was inserted into the right spinal cord at C 4 after performing a laminectomy. The micropipette (75-150 #m tip diameter, 1-2 MQ) was filled with a mixture of 50% of a 2% saline solution and 50% of a 2% Fast blue (FB, Dr. Illing and Co) t6 solution in ethanol (90%). Injection was made by pressure pulses apphed to the same micropipette (Fig. 1A) at the site where an inspiratory activity was recorded (Fig. 1B). To identify the propriobulbar neurons in VRG projecting to expiratory neurons of right cVRG a micropipette was inserted into the caudal medulla at the appropriate coordinates (between 1 and 1.5 mm caudal to zero). For that purpose the head of the animal was ventroflexed 25° to the horizontal plane of Paxinos and Watson 23, and an occipital craniotomy was performed to access the medulla. The micropipette had the same characteristics as those described above, but was filled with a mixture of 50% of the saline solution and 50% of a 2% Diamidino yellow (DY, Dr. Ilhng) 15 solution in ethanol. This micropipette was inserted into the medulla at the appropriate coordinates to find spontaneous expiratory activity (Fig 1B). Injection of DY was made by pressure pulses applied to the same rmcropipette at the site where an expiratory actiwty was recorded (Fig. 1C,D). After withdrawal of the micropipette, the spinal cord and the medulla were protected with a silicone sheet, and the surgical wounds were sutured
H~stology Following a survwal period of 7 days, the animals were deeply
re-anesthetized with sodium pentobarbltal (50 mg/kg 1.p.) and perfused transcardially with 10% formahn in 0.I M phosphate buffer (pH 7.4). The brains and spinal cords were removed and kept overnight m 10% sucrose in 0 1 M phosphate buffer (pH "7 4). They were sectioned (50/~m) on a freezing mlcrotome. Sections were mounted onto gelatin-coated shdes, air dned and coverslipped with DPX. Every third section was counterstained with Neutral red solution for reference purposes. The fluorochrome-labeled neurons were stud~ed under a Zelss axlophot transmitted fluorescence m~croscope equipped wlth a UV-H (365 nm wavelength) filter system The neurons studied in this report were those within NA itself and the surrounding medullary reticular field23, where respiratory neurons have been described by several authors 6'8"29. The selected neurons were photographed using high-speed film (400 ASA) for black and white prints. Most reports on regions of respiratory activity m the rat have used the obex as an anatomic reference point. But there ~s no agreement concerning the location of the obex. For some, ~t is the beginning of the area postrema in the medulla s (interaural -5.30 mm, Bregma -14 30 mm, Paxinos and Watson Atlas23), while for others it is the junction between the central canal of the spinal cord and the IVth ventricle of the medulla 4'6'2s (interaural -4 30 mm, Bregma -13 30 mm, Paxmos and Watson atlas23). The reference point chosen for the present report was the same as that described by EUenberger and Feldman 6, the rostral edge of the area postrema taken as a zero.
RESULTS No double labeled neurons were found.
Proptobulbar neurons D Y was i n j e c t e d in an a r e a of the c V R G w h e r e expiratory neurons were predominantly recorded. The area o v e r which the t r a c e r s p r e a d was always b e t w e e n 1.0 and 1.5 m m c a u d a l to the rostral e d g e of the a r e a p o s t r e m a , within the N A and the c a u d a l v e n t r o l a t e r a l reticular nucleus. T h e t r a c e r n e v e r r e a c h e d the lateral r e t i c u l a r nucleus (Fig. 1 C , D ) . F o l l o w i n g the i n j e c t i o n o f D Y into t h e c V R G (Fig. 1C), l a b e l e d n e u r o n s w e r e f o u n d t h r o u g h o u t the brains t e m , in nuclei o t h e r t h a n t h e r e g i o n of N A and surr o u n d i n g r e t i c u l a r field (Fig. 3). H o w e v e r , c o u n t s of lab e l e d cells w e r e m a d e only within the r e g i o n w h i c h constitutes the V R G (Fig. 2 A ) . R o s t r a l to the i n j e c t i o n site, i.e. at a level b e g i n n i n g 1 m m c a u d a l to the rostral e d g e of the a r e a p o s t r e m a , m o s t l a b e l e d n e u r o n s (average n u m b e r o f cells p e r a n i m a l , n = 682, r a n g e 5 3 2 - 7 9 5 , or 65%) w e r e ipsilateral, and the o t h e r (n = 353, r a n g e 2 5 6 - 3 9 8 , or 35%) w e r e c o n t r a l a t e r a l to the site of injection. A t the level of t h e injection site an a v e r a g e o f 80 contralateral labeled neurons were found (range 65110). C a u d a l to the injection site, an a v e r a g e of 65 ( r a n g e 3 8 - 1 4 5 ) l a b e l e d n e u r o n s w e r e ipsilateral, and an a v e r a g e o f 31.5 (range 11-60) w e r e c o n t r a l a t e r a l , giving a p e r c e n t a g e of 68 and 32, respectively. T h e density of l a b e l e d n e u r o n s c h a n g e d a l o n g the rost r o c a u d a l axis of the V R G . P e a k density of l a b e l e d cells was o b s e r v e d ipsilaterally close to the site o f injection.
167
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Fig. 1. A: low power photomicrograph under transmitted fluorescence microscope of a coronal section of the (34 level of the spinal cord showing the location of the Fast blue injection (arrow) into phrenic motoneurous. Bar: 200/~m. B: above, inspiratory multi-unit activity of the recorded phrenic motoneurons in the spinal cord; below, expiratory multi-unit activity of the recorded cVRG neurons in the medulla. C: low power light photonucrograph of a coronal section of the medulla shovong the locatton of Diamidino yellow injection site (arrow) into the cVRG neurons recorded in B. Bar: 200/~m. D: high power photomicrograph of the injection site showed in C, under transmitted fluorescence microscope. Bar: 50 #m. Abbreviations: IOB, inferior olive nucleus; LRt, lateral reticular nucleus; Sp5C, spinal trigeminal nucleus caudal.
The density of labeled cells at 1 m m rostral to the rostral edge of the area postrema was quite similar on both
sides elsewhere (Fig. 2A). In the V R G an average of 806.5 (range 632-995) labeled n e u r o n s (67%) were yen-
168 tral to NA, and the remaining (33%) were dorsal. Moreover, the distribution of these two subgroups differed along the rostrocaudal axis of NA. Dorsal neurons were more numerous in the most caudal levels of NA (between -1 and -0.5 mm caudal to the rostral edge of the area postrema, Fig. 2D). Ventral neurons were distributed along the whole V R G , primarily ipsilateral to the injection site (Fig. 2E).
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Fig. 3. Camera luclda diagrams of 4 representative coronal sections of the rat medulla (A,B,C and D), showing the location of the 3 different types of labeled somata: cell bodies of propriobulbar projections (filled circles), cell bodies of bulbospinal projections (filled tdangies), and NA motoneurons (filled squares). Labeled neurons not considered in this report were represented by open symbols. 12, hypogiossel nucleus; 12n, hypogiossal nerve; IO, inferior ofive; LRt, lateral reticular nucleus; MLF, medial longitudinal fascicle; py, pyramidal tract; ROb, raphe obscurus nucleus; sp5, spinal trigeminal tract; Sp5C, spinal trigeminal nucleus caudal.
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Fig. 2. Rostrocaudal distribution of each class of labeled neurons. A: labeled cell boches of propriobulbar projections. B: labeled cell bodies of bulbospmal projections. C: labeled cell bodies of motoneurons innervating different supradiaphragmatic structures. D - G : the labeled cell bodies of propriobulbar and bulbospinal projections have been subdivided into two groups according to their locations in the coronal plane with respect to the nucleus amb]guus. Ordinate: mean number of labeled cells (two experiments) in a 100-#m-thick section. Abscissa: rostrocaudal location of the secUons, arrow at zero indicates junction of the central canal of the spinal cord and the IVth ventricle of the medulla, the rostrai edge of the area postrema. Left, caudal; right, rostral.
deus. The injection spreading area was done outside the limits of the phrenic nucleus itself in order to label most neurons whose axons terminate within the phrenic nucleus (Fig. 1A). Labeled neurons appeared throughout the rat medulla, in nuclei other than N A and the surrounding reticular field (Fig. 3), but only labeled ceils within the V R G are described here. An average of 496 (range 314-524) bulbospinal neurons were labeled; this number was less than the propriobulbar neurons labeled. Sixty-five percent of the labeled bulbospinal neurons were ipsilateral to the site of injection (average 322, range 219-368). These counts did not include neurons located at the cVRG which was simultaneously iajected. FB-labeled neurons were distributed along the rostrocaudal axis of NA (Fig. 2B). One half of labeled bulbospinal neurons (average 268, range 195-317) was located ventral to NA, which were more concentrated at the rostral part ipsilaterally (Fig. 2G). The other half of bulbospinal neurons was dorsal to the NA, exhibiting the highest densities of cells at the most caudal levels of NA (Fig 2F). Their dendritic trees were orientated in the coronal plane dorsomedially, towards the vlNTS and ventrolaterally. Motoneurons
N A motoneurons were labeled after the injection of FG into the muscles and nerves as described above (see Materials and Methods). Labeled neurons were less numerous at the caudal pole of NA, increasing in number towards the rostral tip of the nucleus. The highest peak density of labeled neurons was at 1.5 m m rostral to the
169
Fig. 4. Photomicrographs of labeled neurons under transmitted fluorescence microscope taken at the 4 representative levels of the rat medulla shown in the Fig. 3 (A,B,C and D). Diamidino yeUow (DY)-labeled propriobulbar neurons, Fast blue (FB)-labeled bulbospinal neurons and Fluorogold (FO)-labeled motoneurons. A and D: bar, 200 ~m. C and B: bar, 100 ~m. D, dorsal, L, lateral.
170 rostral edge of the area postrema. Rostral to 1.6 mm, the number of labeled Mns decreased until they disappeared abruptly at +2 mm. Several small peak densities within this distribution represented the different subpopulations of neurons constituting the nucleus ambiguus, i.e. laryngeal, pharyngeal and esophageal Mns 2'21, and the vagal parasympathetic neurons outside the limit of NA (Fig. 2C). Their dendritic arborizations were orientated dorsomedially towards the vlNTS and ventrolaterally. Relative locations of propriobulbar neurons, bulbospinal neurons and motoneurons within the VRG Few labeled neurons were present caudal to -1.3 ram. FG-labeled neurons (i.e. motoneurons and parasympathetic neurons) were loosely distributed within the NA. Most of the labeled somata were small and fusiform in shape. These neurons were intermingled with the propriobulbar and bulbospinal neurons (Figs. 3D and 4D). Labeled neurons appeared loosely arranged between 1.3 mm caudal to 0.3 mm rostral to the rostral edge of the area postrema (Figs. 3C and 4C). The great majority of Mns was located at the center of NA, dorsomedial and ventrolateral to them other FO-labeled neurons were found. Labeled Mns had multipolar somata, with the dendrites orientated dorsomedially and ventrolaterally in the coronal plane. Bulbospinal neurons were located dorsal and ventral to the NA, being more scattered dorsally and clustered ventrally. Some were intermingled with propriobulbar cells. A large number of propriobulbar neurons surrounded NA Mns, and were located mostly ipsilateral to the site of injection (Figs. 3C and 4C). NA Mns were scattered in small groups of 3-5 neurons, separated from each other by the cell bodies of propriobulbar and bulbospinal neurons. As shown in the coronal plane, the whole VRG constituted a column of cells orientated dorsomedially, limited ventrally by the lateral reticular nucleus. Towards the solitary tract nucleus, the column becomes thinner (Figs. 3C and 4C). Labeled Mns were prominent between 0.4 and 1 mm rostral to the rostral edge of the area postrema, very dense, and delimited the NA; no other cell type was observed in this region. The 3 types of labeled somata were more closely aggregated here than in the caudal part of the VRG. Very few labeled propriobulbar and bulbospinal neurons were located dorsomedial to the NA. However, ventral to the NA many propriobulbar and bulbospinal neurons, were both intermingled. At this level, the labeled somata of bulbospinal neurons were ventral or ventrolateral to the propriobulbar neurons, and in some examples some Mns were also located between them (Figs. 3B and 4B).
A region of densely packed cell bodies started 1 mm rostral and ended 1.6 mm rostral to the rostral edge of the area postrema. The highest number of Mns, i.e. FGlabeled neurons was found here, thickly clustered. Labeled somata of propriohulbar and a low number of bulbospinal neurons were intermingled, located ventromedially to the NA, but never reacb-ing the paragigantocellular nucleus. Propiobulbar neurons predominated in the center of this surrounding cap. Few labeled neurons were found dorsal to NA in both sides of the medulla (Figs. 3A and 4A). At the rostral tip of the VRG, between 1.6 and 2.1 mm rostral to the rostral edge of the area postrema, the NA overlapped dorsally with the facial nucleus, whose neurons appeared 1.6-1.7 nun rostral to the rostral edge of the area postrema. We found a few cell bodies of propriobulbar neurons surrounding Mns, far beyond the nucleus. No labeled bulbospinal neurons were found. DISCUSSION The present study describes the distribution of neurons within the NA and their surrounding reticular field, corresponding to the so-called VRG 6'29. Within this area we examined neurons with axonal projections to the caudal medulla and neurons with axonal projections to the spinal cord, and their relative location with respect to the cranial motoneurons. Despite the assumed axonal collaterals of BC propriobulbar and bulbospinal interneurons described by other authors in cat and rat (see ref. 7 for review) we were unable to find any double labeling in our study. The present report investigated the organization of the different neuronal populations within the VRG of the rat. Indeed in our study we simultaneously labeled NA Mns, bulbospinal and propriobulbar neurons on both sides of the medulla which revealed their relative location within the ventrolateral medulla, a fact which was not completely documented in previous studies 6'24. Propriobulbar neurons The medullary distribution of propriobulbar neurons was related to 3 respiratory described regions of the VRG: BC, rVRG and cVRG. The greatest density of labeled neurons seen in the BC was in the ipsilateral region from 1 to 1.4 mm rostral to the rostral edge of the area postrema. This predominantly ipsilateral axonal pathway links the rostral and caudal subdivisions of the VRG, which has been demonstrated to contain expiratory neurons 1'1s. This projection was recently demonstrated by spike-triggered averaging 14. Within the VRG inspiratory area i.e. 0.9 caudal to and 0.9 mm rostral to the rostral edge of the area postrema, which showed
171 several kinds of propriobulbar neurons 5'6's, the propriobulbar neurons exhibited several peak densities indicating considerable topographical complexity. The number of labeled neurons rostral to the rostral edge of the area postrema was approximately equal ipsi- and contralaterally to the site of injection. However, caudal to the rostral edge of the area postrema labeled cells appeared more numerous ipsilaterally to the site of injection. This fact could indicate a differential organization in axonal projections from rVRG to cVRG, which could correspond to functional properties which remain to be elucidated. In addition, some labeled neurons appeared contralateral to the injection site providing the link between both sides of cVRG. The examination of the labeled propriobulbar neurons in coronal planes indicated a clear distribution into two subdivisions, one ventral and the other dorsal to the NA. The dorsal subdivision was more dense at the caudal VRG, while the ventral one extended along the whole VRG. This differential organization strongly suggests that these two subdivisions correspond to two neuronal populations having different physiological functions. Indeed, it has been demonstrated that the cardiovascular neurons are concentrated within the ventrolateral part of the medulla, ventral to the NA ~3. Bulbospinal neurons Our results indicate the presence of two clear subpopulations of labeled bulbospinal neurons. One, located between 0.9 and 1.6 mm, corresponding to the BC had primarily ipsilateral spinal projections. This relationship has been demonstrated by electrophysiology, providing the link between the inhibition by the BC expiratory neurons of the phrenic Mns 19. These neurons provide an anatomical link between the BC in the medulla and phrenic motoneurons 6,s. The second was caudal and projected bilaterally to the spinal cord. This second relationship is the anatomical correlate of the main connection between the inspiratory premotor bulbospinal neurons providing excitation to phrenic Mns, as shown in other morphological and electrophysiological studies 4' 22,28
Relative location of propriobulbar neurons, bulbospinal neurons, and motoneurons within the VRG We retrogradely labeled with different fluorochromes 3 types of neurons, i.e. bulbospinal neurons, propriobulbar neurons and motoneurons within the ventrolateral medulla, where they formed a column of cells. As shown by Ellenberger and Feldman 6, these neurons were anatomically organized in subnuclear groups. However,
the present study was somewhat different due to the sites of fluorochrome injections and subsequent neuronal labelings. Indeed, firstly we retrogradely labeled propriobulbar neurons which projected to the cVRG; secondly we examined the neurons of both sides of the medulla projecting to the caudal medulla and the spinal cord, rather than restrict our study to the contralateral side of the medulla, and thirdly, we examined the whole NA, including its semicompact formation, a fact not previously stated 6'24. As in the previous study of Ellenberger and Feldman 6 we showed that the relative subnuclear distribution of the 3 types of neurons is different in the cVRG, rVRG and BC, but the population of propriobulbar neurons we have revealed did not exhibit the same organization since we injected the fluorochrome in sites of the cVRG in which expiratory patterns of discharge were obtained. In the caudal part of the VRG (cVRG), the column of labeled cells was composed of intermingled propriobulbar and bulbospinal neurons surrounding a few vagal motoneurons. In the intermediate part of the column (rVRG) propriobulbar and bulbospinal neurons were less intermingled than in the cVRG, and were located ventromedially to the NA, which appeared there as composed of numerous vagal Mns. Finally at the rostral part of the column (BC), the highest number of vagal and glossopharyngeal Mns was observed, and the propriobulbar and bulbospinal neurons were located ventrally to them. This organization of the 3 types of neurons within the column of labeled cells along the ventrolateral medulla could support the functional diversification shown by the electrophysiologlcal studies. For example, it has been demonstrated in the cat, that excitatory and inhibitory ipsilateral connections between BC expiratory neurons and bulbospinal expiratory neurons of the cVRG 14 and inhibitory (bilateral) connections between BC expiratory neurons and the cVRG and rVRG 1°. Furthermore, the organization of bulbospinal and propriobulbar neurons surrounding the NA Mns in clusters suggested the possibility of axonal collateral connections between them, as demonstrated for bulbospinal inspiratory and inspiratory Mns in the cat 9. This organization could also be the anatomical basis of the neuronal network which makes the coordination between the various functions involving these neurons, i.e. breathing, swallowing, coughing, etc. Acknowledgements. This work was supported by Grants from the C.I.C.Y.T. PB-87 938 and the Junta of Andalueia, and by a FrenchSpamsh 'Action Int6gr6e'. The authors express their appreciation for the technical assistance of Mrs. C. G6rnez-Romb.nand to Prof. S. Iscoe for his critical review of the manuscript.
172 REFERENCES 1 Bianchi, A.L., ~ l i z a t i o n et ~tude des neurones resplratoires buibaires. Mtse en jeu antidromique par stimulation spinale ou vagale, J. Physiol. (Paris), 63 (1971) 5-40. 2 Bieger, D. and Hopkins, D.A., Viscerotopic representation of the upper alimentary tract in the medulla obiongata in the rat: the nucleus ambiguus, J. Comp NeuroL, 262 (1987) 546-562. 3 Bystrzycka, E.K. and Nail, B.S., Brain stem nuclei associated with respiratory, cardiovascular and other autonomic functions. In G. Paxinos (Ed.), The Rat Nervous System. Vol. 2, Hindbrain and Spinal Cord, Vol. 1, Academic, Sidney, 1986, pp. 95-110. 4 Ellenberger, H.H. and Feldman, J.L., Monosynaptic transmission of respiratory drive to phrenic motoneurons from brainstem bulbospinal neurons in rats, J. Comp. Neurol., 269 (1988) 47-57. 5 Ellenberger, H.H. and Feldman, J.L., Bramstem connectmns of the rostral ventral respiratory group of the rat, Brain Research, 513 (1990) 35-42. 6 Ellenberger, H.H. and Feldman, J.L., Subnuclear organization of the lateral tegmental field of the rat. I. Nucleus amblguus and ventral respiratory group, J. Comp. Neurol., 294 (1990) 202-211. 7 Ezure, K., Synaptic connections between medullary respiratory neurons and considerations on the genesis of respiratory rhythm, Progr. Neurobiol., 35 (1990) 429-450. 8 Ezure, K., Manabe, M. and Yamada, H., Distribution of medullary respiratory neurons in the rat, Brain Research, 455 (1988) 262-270. 9 Ezure, K. and Manabe, M., Monosynaptie exotation of medullary inspiratory neurons by bulbospinal inspiratory neurons of the ventral respiratory group in the cat, Exp. Brain Res., 74 (1989) 501-511. 10 Fedorko, L. and Merrill, E.G., Axonal projectmns from the rostral expiratory neurons of the B6tzinger Complex to the medulla and spinal cord in the cat, J. Physiol., 350 (1984) 487496. 11 Feldman, J.L., Loewy, A.D. and Speck, D.E, Projections from the ventral respiratory group to phrenic and intercostal motoneurons in cat: an autoradiographic study, J. Neurosct., 5 (1985) 1993-2000. 12 Gr~lot, L., Barillot, J.C. and Bianchi, A.L., Central distributions of the efferent and afferent components of the pharyngeal branches of the vagus and giossopharyngeal nerves: an HRP study in the cat, Exp. Brain Res., 78 (1989) 327-335. 13 Guyenet, P.G., Darnall, R.A. and Riley, T.A., Rostral ventrolateral medulla and sympathorespiratory integration in rats, Am. J. Physiol. Regul. Integr. Comp. Phystol., 259 (1990) 10631074. 14 Jlang, C. and Lipski, J., Extensive monosynaptlc inhibition of ventral respiratory group neurons by augmenting neurons in the B6tzinger complex in the cat, Exp. Brain Res., 81 (1990) 639648. 15 Keizer, K., Kuypers, H.G.J.M., Huisman, A.M. and Dann,
O., Diarmdino Yellow dlhydrochloride (DY-2HC1): a new fluorescent retrograde neuronal tracer, which migrates only very slowly out of the cell, Exp. Brain Res., 51 (1983) 179-191. 16 Kuypers, H.G.J.M., Bentivoglio, M., Castman-Berrevoets, C.E. and Bharos, T.B., Double retrograde neuronal labeling through divergent axon collaterals, using two fluorescent tracers with the same excitation wavelength which label different features of the cells, Exp. Brain Res., 40 (1980) 383-392. 17 Lawn, A.M., The localization in the nucleus ambiguus of the rabbit, of the cells of origin of motor nerve fibers in the glossopharyngeal nerve and various branches of the vagus nerve by means of retrograde degeneration, J. Comp. Neurol, 127 (1966) 293-306. 18 Merrill, E.G., The lateral respiratory neurons of the medulla: their associations with nucleus ambiguus, nucleus retroambiguahs, the spinal accessory nucleus and the spinal cord, Brain Research, 24 (1970) 11-28. 19 Merrill, E.G. and Fedorko, L., Monosynaptic inhibition of phremc motoneurons: a long descending projection from B0tzinger neurons, J Neurosci., 4 (1984) 2350-2353. 20 N0fiez-Abades, P.A., Portillo, E and P~isaro, R., CharacterisaUon of afferent projections to the nucleus ambiguus of the rat, by means of fluorescent double labeling, J. Anat., 172 (1990) 1-15. 21 N0fiez-Abades, P.A., P~isaro, R. and Bianctu, A.L., Study of the topographical distribution of different populations of motoneurons within rat's nucleus ambiguus, by means of four different fluorochromes, Neurosci. Lett., in press. 22 Onai, T., Saji, M. and Miura, M., Projections of supraspinal structures to the phremc motor nucleus in rats studied by a horseradish peroxidase microinjection method, J. Auton. Nerv. Syst., 21 (1987) 233-240. 23 Paxinos, G. and Watson, C., The Rat Brain m Stereotaxic Coordinates, 2nd edn., Acadermc, Sidney, 1986. 24 Portillo, E and P~isaro, R., Location of bulbospinal neurons and laryngeal motoneurons w~thin the nucleus ambiguus of the rat and cat by means of retrograde fluorescent labeling, J. Anat., 159 (1988) 11-18. 25 Portillo, E and P~saro, R., Location of motoneurons supplying the intrinsic laryngeal muscles of rats. Horseradish peroxidase and fluorescent double-labeling study, Brain Behav. Evol., 32 (1988) 220-225. 26 Saether, K., Hilaire, G. and Monteau, R., Dorsal and ventral respiratory groups in the medulla of the rat, Brain Research, 419 (1987) 87-96. 27 Schmued, L.C. and Fallon, J M., Fluoro-gold: a new fluorescent retrograde axonal tracer with numerous unique properties, Brain Research, 377 (1986) 147-154. 28 Yamada, H., Ezure, K. and Manabe, M., Efferent projections of inspiratory neurons of the ventral respiratory group. A dual labeling study in the rat, Brain Research, 455 (1988) 283-294. 29 Zheng, Y., Barillot, J.C. and Bianchi, A.L., Patterns of membrane potentials and distributions of the medullary respiratory neurons in the decerebrate rat, Brain Research, 546 (1991) 261270.
165
RES 17293
Localization of respiratory bulbospinal and propriobulbar neurons in the region of the nucleus ambiguus of the rat P.A. Nfifiez-Abades 1, R.
P~isaro I a n d A . L . Bianchi 2
1Laboratory of Neuroscience, Department of Animal Physiology and Biology, University of Sevilla, SeviUa (Spain) and ZD#partement de Physiologie et Neurophysiologie, C.N.R.S. URA 205, Facuh# des Sciences et Techniques, Marseille (France)
(Accepted 13 August 1991) Key words: Bttzinger complex; Diamidino yellow; Esophagus; Expiratory neuron; Fast blue; Fluorescent marker; Fluorogold; Nucleus ambiguus; Phrenic nucleus; Rat; Swallowing muscle; Vagal motoneuron; Ventral respiratory group
The location of neurons within the ventral respiratory group (VRG) of rat was mapped following injections of 3 different fluorochrome tracers into different rues known to receive projections from VRG neurons. Injection sites included muscles innervated by the vagus (X) and glossopharyngeal (IX) nerves, and the sites of expiratory activity in the caudal medulla and of inspiratory activity in the spinal cord at the C4 level. Labeling of vagal motoneurous resulting from fluorochrome injections into muscles innervated by X and IX nerves was always ipsilateral to the site of injection. Both propfiobulbar and bulbospinal neurons had primarily ipsilaterai projections. No double-labeled cell bodies were observed. The cell bodies of the 3 types of neurons, propfiobulbar, bulbospinal and vagai/glossopharyngeai,were unevenly distributed along the rostrocaudal axis of the VRG, suggesting a complex mosaic of neurons which regulate respiratory-related functions such as swallowing and vocahzation.
INTRODUCTION The nucleus ambiguus (NA) of the rat consists of a longitudinal cell column of large multipolar neurons lying ventral to the parvocellular reticular nucleus and dorsal to the lateral reticular nucleus. It extends from the facial nucleus as far caudally as the pyramidal decussation 3. Although classically described as being composed of large motoneurons (Mns) which innervate the laryngeal and pharyngeal muscles 17, further studies have demonstrated the existence of a viscerotopic organization TM 12.24. The rostral part contains esophageal Mns (compact formation), the intermediate part contains laryngeal and pharyngeal Mns (semicompact formation), and the caudal part contains laryngeal Mns (loose formation). Ventral and dorsal to this motoneuronal pool, but external to it, lies an aggregation of preganglionic neurons innervating the heart and other supradiaphragmatic structures 2. Morphological and electrophysiological studies have demonstrated the existence of a ventral respiratory group (VRG) of neurons associated with the NA. The V R G contains bulbospinal neurons which provide the premotor control to phrenic, intercostal and abdominal motoneurons 4'11'22. It also contains propriobulbar neurons
interconnected to neurons in other brainstem respiratory groups 5'2°. In the rat V R O , 3 different subgroups of respiratory neurons have been found: (1) the BOtzinger complex (BC), a cluster of neurons in the most rostral part of the V R G located ventromedial to the rostral part of NA, consists primarily of expiratory neurons, (2) the intermediate part of the V R G , rostral to the obex (rVRG), contains mostly inspiratory neurons; and (3) caudal to the obex the caudal division of V R G (cVRG) contains mostly expiratory neurons 6'a'26'29. The anatomical organization of the NA likely provides the structural basis for its functional role in controlling breathing and such other related functions as sneezing, coughing, swallowing, gagging, phonation, and vomiting. We therefore examined the location, morphology, and orientation of the dendritic trees of the 3 different populations of neurons in the N A and the surrounding reticular region which constitute the V R G , i.e. bulbospinal neurons projeering to phrenic Mns, propriobulbar interneurons projecting to expiratory neurons of the cVRG, and vagal and glossopharyngeal neurons projecting to the pharyngeal and laryngeal muscles, and to the esophageal muscles. We investigated both sides of the medulla, allowing us to examine the bilateral organization of the neuronal networks involved in these respiratory-related
Correspondence: R. P~saxo, Laboratorio de Neurociencia, Departemento de Fisiologia y Biologta Animal, Facultad de Biologta, Avda. R¢ina Mercedes 6, 41012-Sevilla, Spain.
166 functions but also to d e t e r m i n e the r e l a t i v e strengths of the ipsilateral and c o n t r a l a t e r a l p r o j e c t i o n s , a topic not e x p l o r e d in p r e v i o u s studies 6'2a. Lastly, t h e s e experim e n t s a l l o w e d us to test for the existence o f possible axonal collateral projections.
MATERIALS AND METHODS Expenments were carried out on 15 albino Wistar rats weighing 250-400 g. The animals were anesthetized with ketamine (50 mg/kg Lp.). In addition, atropine sulfate (0,5 mg/kg i.m.) was admimstered to reduce mucus secretions. Subsequent maintenance doses of ketamine were administered i.m. when needed Rectal temperature was mmntalned at 37-38°C.
Surgical procedures To label the motoneurons of the NA on both sides of medulla, the following muscles and nerves were injected bilaterally: the caudal and medial pharyngeal constrictor muscles, innervated by the pharyngeal branch of the vagus nerve; the stylopharyngeal muscle innervated by the glossopharyngeal nerve (IX); the cricothyroid muscle innervated by the superior laryngeal nerve, and cervical vagus nerve (X) which innervates through the recurrent laryngeal nerve, the posterior cricoarytenoid, lateral cricoarytenoid and thyroarytenoid muscles of the larynx, and the esophagus. Injections of the tracers were made through micropipettes attached to a pressure injection apparatus (Picospritzer) and under direct visual observation using a Zeiss dissecting microscope. The microplpettes (100-200 #m tip diameter) were filled with a 2% fluorogold solution made m sahne (FG, Fluorochrome Inc., Englewood Co.) 27 For injections into muscles, several injections were made until the muscles lost their color. In nerves, injections were made at low pressures after the micropipette tip had been inserted through the perineurium into the nerves. Following injections, the animals were allowed to recover. AmpieiUin (10,000 IU i.m.) was administered Three days later, the animals were anesthetized again as previously described. We monitored central respiratory activity, by means of two recording electrodes (insulated silver wire, 200/tm diameter) which were inserted into the diaphragm from the abdominal side. The signal was amplified and displayed on an oscilloscope. In order to localize bulbospinal neurons in the VRG projecting to the phrenic nucleus, we recorded activity of the phremc motoneurons via a micropipette which was inserted into the right spinal cord at C 4 after performing a laminectomy. The micropipette (75-150 #m tip diameter, 1-2 MQ) was filled with a mixture of 50% of a 2% saline solution and 50% of a 2% Fast blue (FB, Dr. Illing and Co) t6 solution in ethanol (90%). Injection was made by pressure pulses apphed to the same micropipette (Fig. 1A) at the site where an inspiratory activity was recorded (Fig. 1B). To identify the propriobulbar neurons in VRG projecting to expiratory neurons of right cVRG a micropipette was inserted into the caudal medulla at the appropriate coordinates (between 1 and 1.5 mm caudal to zero). For that purpose the head of the animal was ventroflexed 25° to the horizontal plane of Paxinos and Watson 23, and an occipital craniotomy was performed to access the medulla. The micropipette had the same characteristics as those described above, but was filled with a mixture of 50% of the saline solution and 50% of a 2% Diamidino yellow (DY, Dr. Ilhng) 15 solution in ethanol. This micropipette was inserted into the medulla at the appropriate coordinates to find spontaneous expiratory activity (Fig 1B). Injection of DY was made by pressure pulses applied to the same rmcropipette at the site where an expiratory actiwty was recorded (Fig. 1C,D). After withdrawal of the micropipette, the spinal cord and the medulla were protected with a silicone sheet, and the surgical wounds were sutured
H~stology Following a survwal period of 7 days, the animals were deeply
re-anesthetized with sodium pentobarbltal (50 mg/kg 1.p.) and perfused transcardially with 10% formahn in 0.I M phosphate buffer (pH 7.4). The brains and spinal cords were removed and kept overnight m 10% sucrose in 0 1 M phosphate buffer (pH "7 4). They were sectioned (50/~m) on a freezing mlcrotome. Sections were mounted onto gelatin-coated shdes, air dned and coverslipped with DPX. Every third section was counterstained with Neutral red solution for reference purposes. The fluorochrome-labeled neurons were stud~ed under a Zelss axlophot transmitted fluorescence m~croscope equipped wlth a UV-H (365 nm wavelength) filter system The neurons studied in this report were those within NA itself and the surrounding medullary reticular field23, where respiratory neurons have been described by several authors 6'8"29. The selected neurons were photographed using high-speed film (400 ASA) for black and white prints. Most reports on regions of respiratory activity m the rat have used the obex as an anatomic reference point. But there ~s no agreement concerning the location of the obex. For some, ~t is the beginning of the area postrema in the medulla s (interaural -5.30 mm, Bregma -14 30 mm, Paxinos and Watson Atlas23), while for others it is the junction between the central canal of the spinal cord and the IVth ventricle of the medulla 4'6'2s (interaural -4 30 mm, Bregma -13 30 mm, Paxmos and Watson atlas23). The reference point chosen for the present report was the same as that described by EUenberger and Feldman 6, the rostral edge of the area postrema taken as a zero.
RESULTS No double labeled neurons were found.
Proptobulbar neurons D Y was i n j e c t e d in an a r e a of the c V R G w h e r e expiratory neurons were predominantly recorded. The area o v e r which the t r a c e r s p r e a d was always b e t w e e n 1.0 and 1.5 m m c a u d a l to the rostral e d g e of the a r e a p o s t r e m a , within the N A and the c a u d a l v e n t r o l a t e r a l reticular nucleus. T h e t r a c e r n e v e r r e a c h e d the lateral r e t i c u l a r nucleus (Fig. 1 C , D ) . F o l l o w i n g the i n j e c t i o n o f D Y into t h e c V R G (Fig. 1C), l a b e l e d n e u r o n s w e r e f o u n d t h r o u g h o u t the brains t e m , in nuclei o t h e r t h a n t h e r e g i o n of N A and surr o u n d i n g r e t i c u l a r field (Fig. 3). H o w e v e r , c o u n t s of lab e l e d cells w e r e m a d e only within the r e g i o n w h i c h constitutes the V R G (Fig. 2 A ) . R o s t r a l to the i n j e c t i o n site, i.e. at a level b e g i n n i n g 1 m m c a u d a l to the rostral e d g e of the a r e a p o s t r e m a , m o s t l a b e l e d n e u r o n s (average n u m b e r o f cells p e r a n i m a l , n = 682, r a n g e 5 3 2 - 7 9 5 , or 65%) w e r e ipsilateral, and the o t h e r (n = 353, r a n g e 2 5 6 - 3 9 8 , or 35%) w e r e c o n t r a l a t e r a l to the site of injection. A t the level of t h e injection site an a v e r a g e o f 80 contralateral labeled neurons were found (range 65110). C a u d a l to the injection site, an a v e r a g e of 65 ( r a n g e 3 8 - 1 4 5 ) l a b e l e d n e u r o n s w e r e ipsilateral, and an a v e r a g e o f 31.5 (range 11-60) w e r e c o n t r a l a t e r a l , giving a p e r c e n t a g e of 68 and 32, respectively. T h e density of l a b e l e d n e u r o n s c h a n g e d a l o n g the rost r o c a u d a l axis of the V R G . P e a k density of l a b e l e d cells was o b s e r v e d ipsilaterally close to the site o f injection.
167
Inspiratory Multi-unit Activity
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Fig. 1. A: low power photomicrograph under transmitted fluorescence microscope of a coronal section of the (34 level of the spinal cord showing the location of the Fast blue injection (arrow) into phrenic motoneurous. Bar: 200/~m. B: above, inspiratory multi-unit activity of the recorded phrenic motoneurons in the spinal cord; below, expiratory multi-unit activity of the recorded cVRG neurons in the medulla. C: low power light photonucrograph of a coronal section of the medulla shovong the locatton of Diamidino yellow injection site (arrow) into the cVRG neurons recorded in B. Bar: 200/~m. D: high power photomicrograph of the injection site showed in C, under transmitted fluorescence microscope. Bar: 50 #m. Abbreviations: IOB, inferior olive nucleus; LRt, lateral reticular nucleus; Sp5C, spinal trigeminal nucleus caudal.
The density of labeled cells at 1 m m rostral to the rostral edge of the area postrema was quite similar on both
sides elsewhere (Fig. 2A). In the V R G an average of 806.5 (range 632-995) labeled n e u r o n s (67%) were yen-
168 tral to NA, and the remaining (33%) were dorsal. Moreover, the distribution of these two subgroups differed along the rostrocaudal axis of NA. Dorsal neurons were more numerous in the most caudal levels of NA (between -1 and -0.5 mm caudal to the rostral edge of the area postrema, Fig. 2D). Ventral neurons were distributed along the whole V R G , primarily ipsilateral to the injection site (Fig. 2E).
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Fig. 3. Camera luclda diagrams of 4 representative coronal sections of the rat medulla (A,B,C and D), showing the location of the 3 different types of labeled somata: cell bodies of propriobulbar projections (filled circles), cell bodies of bulbospinal projections (filled tdangies), and NA motoneurons (filled squares). Labeled neurons not considered in this report were represented by open symbols. 12, hypogiossel nucleus; 12n, hypogiossal nerve; IO, inferior ofive; LRt, lateral reticular nucleus; MLF, medial longitudinal fascicle; py, pyramidal tract; ROb, raphe obscurus nucleus; sp5, spinal trigeminal tract; Sp5C, spinal trigeminal nucleus caudal.
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Fig. 2. Rostrocaudal distribution of each class of labeled neurons. A: labeled cell boches of propriobulbar projections. B: labeled cell bodies of bulbospmal projections. C: labeled cell bodies of motoneurons innervating different supradiaphragmatic structures. D - G : the labeled cell bodies of propriobulbar and bulbospinal projections have been subdivided into two groups according to their locations in the coronal plane with respect to the nucleus amb]guus. Ordinate: mean number of labeled cells (two experiments) in a 100-#m-thick section. Abscissa: rostrocaudal location of the secUons, arrow at zero indicates junction of the central canal of the spinal cord and the IVth ventricle of the medulla, the rostrai edge of the area postrema. Left, caudal; right, rostral.
deus. The injection spreading area was done outside the limits of the phrenic nucleus itself in order to label most neurons whose axons terminate within the phrenic nucleus (Fig. 1A). Labeled neurons appeared throughout the rat medulla, in nuclei other than N A and the surrounding reticular field (Fig. 3), but only labeled ceils within the V R G are described here. An average of 496 (range 314-524) bulbospinal neurons were labeled; this number was less than the propriobulbar neurons labeled. Sixty-five percent of the labeled bulbospinal neurons were ipsilateral to the site of injection (average 322, range 219-368). These counts did not include neurons located at the cVRG which was simultaneously iajected. FB-labeled neurons were distributed along the rostrocaudal axis of NA (Fig. 2B). One half of labeled bulbospinal neurons (average 268, range 195-317) was located ventral to NA, which were more concentrated at the rostral part ipsilaterally (Fig. 2G). The other half of bulbospinal neurons was dorsal to the NA, exhibiting the highest densities of cells at the most caudal levels of NA (Fig 2F). Their dendritic trees were orientated in the coronal plane dorsomedially, towards the vlNTS and ventrolaterally. Motoneurons
N A motoneurons were labeled after the injection of FG into the muscles and nerves as described above (see Materials and Methods). Labeled neurons were less numerous at the caudal pole of NA, increasing in number towards the rostral tip of the nucleus. The highest peak density of labeled neurons was at 1.5 m m rostral to the
169
Fig. 4. Photomicrographs of labeled neurons under transmitted fluorescence microscope taken at the 4 representative levels of the rat medulla shown in the Fig. 3 (A,B,C and D). Diamidino yeUow (DY)-labeled propriobulbar neurons, Fast blue (FB)-labeled bulbospinal neurons and Fluorogold (FO)-labeled motoneurons. A and D: bar, 200 ~m. C and B: bar, 100 ~m. D, dorsal, L, lateral.
170 rostral edge of the area postrema. Rostral to 1.6 mm, the number of labeled Mns decreased until they disappeared abruptly at +2 mm. Several small peak densities within this distribution represented the different subpopulations of neurons constituting the nucleus ambiguus, i.e. laryngeal, pharyngeal and esophageal Mns 2'21, and the vagal parasympathetic neurons outside the limit of NA (Fig. 2C). Their dendritic arborizations were orientated dorsomedially towards the vlNTS and ventrolaterally. Relative locations of propriobulbar neurons, bulbospinal neurons and motoneurons within the VRG Few labeled neurons were present caudal to -1.3 ram. FG-labeled neurons (i.e. motoneurons and parasympathetic neurons) were loosely distributed within the NA. Most of the labeled somata were small and fusiform in shape. These neurons were intermingled with the propriobulbar and bulbospinal neurons (Figs. 3D and 4D). Labeled neurons appeared loosely arranged between 1.3 mm caudal to 0.3 mm rostral to the rostral edge of the area postrema (Figs. 3C and 4C). The great majority of Mns was located at the center of NA, dorsomedial and ventrolateral to them other FO-labeled neurons were found. Labeled Mns had multipolar somata, with the dendrites orientated dorsomedially and ventrolaterally in the coronal plane. Bulbospinal neurons were located dorsal and ventral to the NA, being more scattered dorsally and clustered ventrally. Some were intermingled with propriobulbar cells. A large number of propriobulbar neurons surrounded NA Mns, and were located mostly ipsilateral to the site of injection (Figs. 3C and 4C). NA Mns were scattered in small groups of 3-5 neurons, separated from each other by the cell bodies of propriobulbar and bulbospinal neurons. As shown in the coronal plane, the whole VRG constituted a column of cells orientated dorsomedially, limited ventrally by the lateral reticular nucleus. Towards the solitary tract nucleus, the column becomes thinner (Figs. 3C and 4C). Labeled Mns were prominent between 0.4 and 1 mm rostral to the rostral edge of the area postrema, very dense, and delimited the NA; no other cell type was observed in this region. The 3 types of labeled somata were more closely aggregated here than in the caudal part of the VRG. Very few labeled propriobulbar and bulbospinal neurons were located dorsomedial to the NA. However, ventral to the NA many propriobulbar and bulbospinal neurons, were both intermingled. At this level, the labeled somata of bulbospinal neurons were ventral or ventrolateral to the propriobulbar neurons, and in some examples some Mns were also located between them (Figs. 3B and 4B).
A region of densely packed cell bodies started 1 mm rostral and ended 1.6 mm rostral to the rostral edge of the area postrema. The highest number of Mns, i.e. FGlabeled neurons was found here, thickly clustered. Labeled somata of propriohulbar and a low number of bulbospinal neurons were intermingled, located ventromedially to the NA, but never reacb-ing the paragigantocellular nucleus. Propiobulbar neurons predominated in the center of this surrounding cap. Few labeled neurons were found dorsal to NA in both sides of the medulla (Figs. 3A and 4A). At the rostral tip of the VRG, between 1.6 and 2.1 mm rostral to the rostral edge of the area postrema, the NA overlapped dorsally with the facial nucleus, whose neurons appeared 1.6-1.7 nun rostral to the rostral edge of the area postrema. We found a few cell bodies of propriobulbar neurons surrounding Mns, far beyond the nucleus. No labeled bulbospinal neurons were found. DISCUSSION The present study describes the distribution of neurons within the NA and their surrounding reticular field, corresponding to the so-called VRG 6'29. Within this area we examined neurons with axonal projections to the caudal medulla and neurons with axonal projections to the spinal cord, and their relative location with respect to the cranial motoneurons. Despite the assumed axonal collaterals of BC propriobulbar and bulbospinal interneurons described by other authors in cat and rat (see ref. 7 for review) we were unable to find any double labeling in our study. The present report investigated the organization of the different neuronal populations within the VRG of the rat. Indeed in our study we simultaneously labeled NA Mns, bulbospinal and propriobulbar neurons on both sides of the medulla which revealed their relative location within the ventrolateral medulla, a fact which was not completely documented in previous studies 6'24. Propriobulbar neurons The medullary distribution of propriobulbar neurons was related to 3 respiratory described regions of the VRG: BC, rVRG and cVRG. The greatest density of labeled neurons seen in the BC was in the ipsilateral region from 1 to 1.4 mm rostral to the rostral edge of the area postrema. This predominantly ipsilateral axonal pathway links the rostral and caudal subdivisions of the VRG, which has been demonstrated to contain expiratory neurons 1'1s. This projection was recently demonstrated by spike-triggered averaging 14. Within the VRG inspiratory area i.e. 0.9 caudal to and 0.9 mm rostral to the rostral edge of the area postrema, which showed
171 several kinds of propriobulbar neurons 5'6's, the propriobulbar neurons exhibited several peak densities indicating considerable topographical complexity. The number of labeled neurons rostral to the rostral edge of the area postrema was approximately equal ipsi- and contralaterally to the site of injection. However, caudal to the rostral edge of the area postrema labeled cells appeared more numerous ipsilaterally to the site of injection. This fact could indicate a differential organization in axonal projections from rVRG to cVRG, which could correspond to functional properties which remain to be elucidated. In addition, some labeled neurons appeared contralateral to the injection site providing the link between both sides of cVRG. The examination of the labeled propriobulbar neurons in coronal planes indicated a clear distribution into two subdivisions, one ventral and the other dorsal to the NA. The dorsal subdivision was more dense at the caudal VRG, while the ventral one extended along the whole VRG. This differential organization strongly suggests that these two subdivisions correspond to two neuronal populations having different physiological functions. Indeed, it has been demonstrated that the cardiovascular neurons are concentrated within the ventrolateral part of the medulla, ventral to the NA ~3. Bulbospinal neurons Our results indicate the presence of two clear subpopulations of labeled bulbospinal neurons. One, located between 0.9 and 1.6 mm, corresponding to the BC had primarily ipsilateral spinal projections. This relationship has been demonstrated by electrophysiology, providing the link between the inhibition by the BC expiratory neurons of the phrenic Mns 19. These neurons provide an anatomical link between the BC in the medulla and phrenic motoneurons 6,s. The second was caudal and projected bilaterally to the spinal cord. This second relationship is the anatomical correlate of the main connection between the inspiratory premotor bulbospinal neurons providing excitation to phrenic Mns, as shown in other morphological and electrophysiological studies 4' 22,28
Relative location of propriobulbar neurons, bulbospinal neurons, and motoneurons within the VRG We retrogradely labeled with different fluorochromes 3 types of neurons, i.e. bulbospinal neurons, propriobulbar neurons and motoneurons within the ventrolateral medulla, where they formed a column of cells. As shown by Ellenberger and Feldman 6, these neurons were anatomically organized in subnuclear groups. However,
the present study was somewhat different due to the sites of fluorochrome injections and subsequent neuronal labelings. Indeed, firstly we retrogradely labeled propriobulbar neurons which projected to the cVRG; secondly we examined the neurons of both sides of the medulla projecting to the caudal medulla and the spinal cord, rather than restrict our study to the contralateral side of the medulla, and thirdly, we examined the whole NA, including its semicompact formation, a fact not previously stated 6'24. As in the previous study of Ellenberger and Feldman 6 we showed that the relative subnuclear distribution of the 3 types of neurons is different in the cVRG, rVRG and BC, but the population of propriobulbar neurons we have revealed did not exhibit the same organization since we injected the fluorochrome in sites of the cVRG in which expiratory patterns of discharge were obtained. In the caudal part of the VRG (cVRG), the column of labeled cells was composed of intermingled propriobulbar and bulbospinal neurons surrounding a few vagal motoneurons. In the intermediate part of the column (rVRG) propriobulbar and bulbospinal neurons were less intermingled than in the cVRG, and were located ventromedially to the NA, which appeared there as composed of numerous vagal Mns. Finally at the rostral part of the column (BC), the highest number of vagal and glossopharyngeal Mns was observed, and the propriobulbar and bulbospinal neurons were located ventrally to them. This organization of the 3 types of neurons within the column of labeled cells along the ventrolateral medulla could support the functional diversification shown by the electrophysiologlcal studies. For example, it has been demonstrated in the cat, that excitatory and inhibitory ipsilateral connections between BC expiratory neurons and bulbospinal expiratory neurons of the cVRG 14 and inhibitory (bilateral) connections between BC expiratory neurons and the cVRG and rVRG 1°. Furthermore, the organization of bulbospinal and propriobulbar neurons surrounding the NA Mns in clusters suggested the possibility of axonal collateral connections between them, as demonstrated for bulbospinal inspiratory and inspiratory Mns in the cat 9. This organization could also be the anatomical basis of the neuronal network which makes the coordination between the various functions involving these neurons, i.e. breathing, swallowing, coughing, etc. Acknowledgements. This work was supported by Grants from the C.I.C.Y.T. PB-87 938 and the Junta of Andalueia, and by a FrenchSpamsh 'Action Int6gr6e'. The authors express their appreciation for the technical assistance of Mrs. C. G6rnez-Romb.nand to Prof. S. Iscoe for his critical review of the manuscript.
172 REFERENCES 1 Bianchi, A.L., ~ l i z a t i o n et ~tude des neurones resplratoires buibaires. Mtse en jeu antidromique par stimulation spinale ou vagale, J. Physiol. (Paris), 63 (1971) 5-40. 2 Bieger, D. and Hopkins, D.A., Viscerotopic representation of the upper alimentary tract in the medulla obiongata in the rat: the nucleus ambiguus, J. Comp NeuroL, 262 (1987) 546-562. 3 Bystrzycka, E.K. and Nail, B.S., Brain stem nuclei associated with respiratory, cardiovascular and other autonomic functions. In G. Paxinos (Ed.), The Rat Nervous System. Vol. 2, Hindbrain and Spinal Cord, Vol. 1, Academic, Sidney, 1986, pp. 95-110. 4 Ellenberger, H.H. and Feldman, J.L., Monosynaptic transmission of respiratory drive to phrenic motoneurons from brainstem bulbospinal neurons in rats, J. Comp. Neurol., 269 (1988) 47-57. 5 Ellenberger, H.H. and Feldman, J.L., Bramstem connectmns of the rostral ventral respiratory group of the rat, Brain Research, 513 (1990) 35-42. 6 Ellenberger, H.H. and Feldman, J.L., Subnuclear organization of the lateral tegmental field of the rat. I. Nucleus amblguus and ventral respiratory group, J. Comp. Neurol., 294 (1990) 202-211. 7 Ezure, K., Synaptic connections between medullary respiratory neurons and considerations on the genesis of respiratory rhythm, Progr. Neurobiol., 35 (1990) 429-450. 8 Ezure, K., Manabe, M. and Yamada, H., Distribution of medullary respiratory neurons in the rat, Brain Research, 455 (1988) 262-270. 9 Ezure, K. and Manabe, M., Monosynaptie exotation of medullary inspiratory neurons by bulbospinal inspiratory neurons of the ventral respiratory group in the cat, Exp. Brain Res., 74 (1989) 501-511. 10 Fedorko, L. and Merrill, E.G., Axonal projectmns from the rostral expiratory neurons of the B6tzinger Complex to the medulla and spinal cord in the cat, J. Physiol., 350 (1984) 487496. 11 Feldman, J.L., Loewy, A.D. and Speck, D.E, Projections from the ventral respiratory group to phrenic and intercostal motoneurons in cat: an autoradiographic study, J. Neurosct., 5 (1985) 1993-2000. 12 Gr~lot, L., Barillot, J.C. and Bianchi, A.L., Central distributions of the efferent and afferent components of the pharyngeal branches of the vagus and giossopharyngeal nerves: an HRP study in the cat, Exp. Brain Res., 78 (1989) 327-335. 13 Guyenet, P.G., Darnall, R.A. and Riley, T.A., Rostral ventrolateral medulla and sympathorespiratory integration in rats, Am. J. Physiol. Regul. Integr. Comp. Phystol., 259 (1990) 10631074. 14 Jlang, C. and Lipski, J., Extensive monosynaptlc inhibition of ventral respiratory group neurons by augmenting neurons in the B6tzinger complex in the cat, Exp. Brain Res., 81 (1990) 639648. 15 Keizer, K., Kuypers, H.G.J.M., Huisman, A.M. and Dann,
O., Diarmdino Yellow dlhydrochloride (DY-2HC1): a new fluorescent retrograde neuronal tracer, which migrates only very slowly out of the cell, Exp. Brain Res., 51 (1983) 179-191. 16 Kuypers, H.G.J.M., Bentivoglio, M., Castman-Berrevoets, C.E. and Bharos, T.B., Double retrograde neuronal labeling through divergent axon collaterals, using two fluorescent tracers with the same excitation wavelength which label different features of the cells, Exp. Brain Res., 40 (1980) 383-392. 17 Lawn, A.M., The localization in the nucleus ambiguus of the rabbit, of the cells of origin of motor nerve fibers in the glossopharyngeal nerve and various branches of the vagus nerve by means of retrograde degeneration, J. Comp. Neurol, 127 (1966) 293-306. 18 Merrill, E.G., The lateral respiratory neurons of the medulla: their associations with nucleus ambiguus, nucleus retroambiguahs, the spinal accessory nucleus and the spinal cord, Brain Research, 24 (1970) 11-28. 19 Merrill, E.G. and Fedorko, L., Monosynaptic inhibition of phremc motoneurons: a long descending projection from B0tzinger neurons, J Neurosci., 4 (1984) 2350-2353. 20 N0fiez-Abades, P.A., Portillo, E and P~isaro, R., CharacterisaUon of afferent projections to the nucleus ambiguus of the rat, by means of fluorescent double labeling, J. Anat., 172 (1990) 1-15. 21 N0fiez-Abades, P.A., P~isaro, R. and Bianctu, A.L., Study of the topographical distribution of different populations of motoneurons within rat's nucleus ambiguus, by means of four different fluorochromes, Neurosci. Lett., in press. 22 Onai, T., Saji, M. and Miura, M., Projections of supraspinal structures to the phremc motor nucleus in rats studied by a horseradish peroxidase microinjection method, J. Auton. Nerv. Syst., 21 (1987) 233-240. 23 Paxinos, G. and Watson, C., The Rat Brain m Stereotaxic Coordinates, 2nd edn., Acadermc, Sidney, 1986. 24 Portillo, E and P~isaro, R., Location of bulbospinal neurons and laryngeal motoneurons w~thin the nucleus ambiguus of the rat and cat by means of retrograde fluorescent labeling, J. Anat., 159 (1988) 11-18. 25 Portillo, E and P~saro, R., Location of motoneurons supplying the intrinsic laryngeal muscles of rats. Horseradish peroxidase and fluorescent double-labeling study, Brain Behav. Evol., 32 (1988) 220-225. 26 Saether, K., Hilaire, G. and Monteau, R., Dorsal and ventral respiratory groups in the medulla of the rat, Brain Research, 419 (1987) 87-96. 27 Schmued, L.C. and Fallon, J M., Fluoro-gold: a new fluorescent retrograde axonal tracer with numerous unique properties, Brain Research, 377 (1986) 147-154. 28 Yamada, H., Ezure, K. and Manabe, M., Efferent projections of inspiratory neurons of the ventral respiratory group. A dual labeling study in the rat, Brain Research, 455 (1988) 283-294. 29 Zheng, Y., Barillot, J.C. and Bianchi, A.L., Patterns of membrane potentials and distributions of the medullary respiratory neurons in the decerebrate rat, Brain Research, 546 (1991) 261270.
