Projections of The Caudal Ventrolateral Medulla

to The Phrenic Nucleus in The Rat

S. G. Patrick Hardy, John G. Horecky, and Kacy G. Presley

Departments of Physical Therapy and Anatomy,

University of Mississippi Medical Center, Jackson, MS 39216

Background: The caudal ventrolateral medulla (CVLM) contains neurons (composing much of the ventral respiratory group) that project to the phrenic nucleus and thereby modulate respiratory functions. However, descriptions of these medullophrenic pathways differ considerably. Consequently, the purpose of this study was to reinvestigate the projections from the CVLM to the phrenic nucleus. Methods: To orthogradely label the medullophrenic projections, either biotinylated dextran amine or lectin conjugated horseradish peroxidase was injected into the CVLM of rats. To retrogradely label the phrenic motor neurons, cholera toxin subunit B-horseradish peroxidase was injected into the diaphragm. Axons descending from the CVLM and their terminations in the phrenic motor nucleus were subsequently plotted. Results: Axons from the CVLM were observed to descend bilaterally, but with an ipsilateral predominance, through the ventral and lateral funiculi of the cervical spinal cord. Decussations were observed within the rostral and caudal medulla, as well as within the spinal cord. Terminations within the phrenic nuclei were observed bilaterally, although the projections to the contralateral phrenic nucleus were somewhat heavier. The close apposition of labeled terminals to labeled motor neurons indicates that the labeled axons may contact the phrenic motor neurons directly. Conclusions: Because the CVLM projects to the phrenic nuclei bilaterally, it is suggested that each CVLM has the potential to influence both phrenic nuclei and subsequently both sides of the diaphragm. However, the fact that the CVLM projects more heavily to the contralateral phrenic nucleus tends to indicate that (in the rat at least) the CVLM-phrenic influences are primarily exerted via the contralateral phrenic nucleus and diaphragm.

Key Words: nucleus ambiguus, ventral respiratory group, biotinylated dextran amine, cholera toxin subunit B-horseradish peroxidase, lectin conjugated horseradish peroxidase

Abbreviations: C nucleus cuneatus; CC central canal; G nucleus gracilis; IO inferior olivary nucleus; LRm lateral reticular nucleus, pars magnocellularis; MV medial vestibular nucleus; NA nucleus ambiguus; NTS nucleus of the solitary tract; P pyramid; Pd pyramidal decussation; PH nucleus prepositus hypoglossi; Rf retrofacial nucleus; ST spinal trigeminal nucleus; X vagal motor nucleus; XII hypoglossal nucleus

Two regions within the medulla (oblongata), functioning to influence respiration, have been studied extensively (see Long and Duffin, 1984 and Feldman, 1986 for reviews). These functionally-defined medullary sites are generally referred to as the dorsal and ventral respiratory groups. Each of these groups corresponds rather imprecisely with known neuroanatomical landmarks. The dorsal respiratory group, composed of inspiratory neurons, is located ventrolateral to the solitary tract (Merrill, 1970; Hilaire and Monteau 1976; Long and Duffin, 1984; Saether et al., 1987). The ventral respiratory group, composed of both inspiratory and expiratory neurons, extends through the ventrolateral medulla (see Feldman, 1986 for review). For the most part the ventral respiratory group lies within, and immediately ventrolateral to, the nucleus ambiguus (Merrill, 1970; Hilaire and Monteau, 1976; Long and Duffin, 1984; Onai et al., 1987; Saether et al., 1987; Ezure et al., 1988; Ellenberger et al., 1990a; Portillo and Nunez-Abades, 1992). As such, the ventral respiratory group is largely contained within the caudal ventrolateral medulla (CVLM) located at, or caudal to, the level of the obex (Onai et al., 1987; Portillo and Nunez-Abades, 1992).

In physiological studies it has been revealed that many neurons of the ventral respiratory group project axons into the cervical spinal cord where they influence the output of the phrenic nucleus (i.e., the phrenic nerve innervation of the diaphragm) and hence modulate inspiration (Merrill, 1970; Hilaire and Monteau, 1976). Supporting this finding, it has been found in anatomical studies involving cats (Feldman et al., 1985; Portillo et al., 1986; Holstege, 1989), rabbits (Ellenberger et al., 1990a) and rats (Onai et al., 1987; Ellenberger and Feldman, 1988; Yamada et al., 1988; Ellenberger et al., 1990b; Portillo and Nunez-Abades, 1992) that neurons of the CVLM (including those within the nucleus ambiguus) have axonal projections to the inspiratory neurons of the phrenic nucleus (Ellenberger and Feldman, 1988; Yamada et al., 1988). In view of these reports, it is generally uncontested that many neurons of the ventral respiratory group project to and influence (either directly or indirectly) the functions of the phrenic nucleus. However, descriptions of the descending projections from the CVLM to the phrenic nucleus tend to vary somewhat with each account. For example, in the cat, this projection has been described as descending through the contralateral ventral funiculus and terminating almost exclusively within the contralateral phrenic nucleus (Merrill, 1970); whereas, others (Feldman et al., 1985) have described the projection in the cat as descending bilaterally through the ventral portion of the lateral funiculus and terminating bilaterally (but having a contralateral predominance) upon the phrenic nuclei. In the rat, the projection from the CVLM to the phrenic nucleus has been described as descending through both the ventral and lateral funiculi and terminating primarily upon the contralateral phrenic nucleus (Ellenberger and Feldman, 1988); whereas, others (Yamada et al., 1988) have described the projection as coursing through the anterior portion of the lateral funiculus and terminating primarily upon the ipsilateral phrenic nucleus.

The purpose of this study was to re-evaluate the projections from the ventral respiratory group, as centered within the CVLM (Merrill, 1970; Hilaire and Monteau, 1976; Long and Duffin, 1984; Saether et al., 1987), to the phrenic motor neurons of the rat. This was accomplished by using orthogradely-transported tracers to label the CVLM-phrenic terminals, and a retrogradely-transported tracer (injected into the diaphragm) to label phrenic motor neurons. Accordingly, the medullary projections to the phrenic nuclei, with respect to their trajectory and sites of termination, were observed.

MATERIALS AND METHODS

A total of 25 Sprague-Dawley rats (Harlan), weighing 300­350 g, were used in this study. In 20 of these animals, biotinylated dextran amine (BDA; 10% in phosphate buffer; Molecular Probes) was injected into the CVLM; whereas, in 5 animals lectin conjugated horseradish peroxidase (WGA-HRP; 1% in distilled water; Sigma) was injected into this same region. The WGA-HRP injections were made for control purposes because, unlike BDA (Brandt and Apkarian, 1992), WGA-HRP is not taken up by unbroken axons of passage (Zaborszky and Heimer, 1989). In 3 of the BDA cases, injections of a highly sensitive (Trojanowski et al., 1982; Sawchenko and Gerfen, 1985), retrogradely-transported tracer known as cholera toxin subunit B-horseradish peroxidase (CTb-HRP; 1% in distilled water; Sigma) were made into the diaphragm. These latter injections served to retrogradely label phrenic motor neurons. By injecting the diaphragm, rather than the phrenic nerve as performed in previous studies (Ellenberger and Feldman, 1988; Yamada et al., 1988), possible leakage of the tracer onto the infrahyoid and scalene muscles (which are innervated by cervical levels of the spinal cord) was prevented. Each of these injections is further described below.

Animal procedures were performed in accordance with NIH guidelines and institutional regulations for animal care and use. Prior to surgery, each animal was anesthetized with chloral hydrate (400 mg/kg, i.p.), and during the surgery the level of anesthesia was maintained at a point where the corneal and cutaneous reflexes were completely absent.

Medullary Injections--In each anesthetized rat, a craniotomy (2­3 mm diameter) was made into either the left or right side of the posterior skull surface, whereupon the tip (20­40 µm diameter) of a glass micropipette, containing either BDA or WGA-HRP, was stereotaxically positioned within either the left or right CVLM. The tracers were then pressure injected (70­180 nl for BDA; 30­50 nl for WGA-HRP) over a 15­20 min period.

Diaphragm Injections--Injections of CTb-HRP were made into the diaphragm in 3 rats that previously had BDA injected into the contralateral medulla. These injections were made 7 days after the BDA injection (and 3 days prior to sacrifice), in animals which had been reanesthetized with chloral hydrate. The diaphragm was exposed after making an incision (approx. 2 cm) through the anterior abdominal wall. Using sterile sponges, the liver was then depressed in order to reveal the diaphragm. Thereafter, CTb-HRP was injected (1­2 µl) into the lateral edge of the diaphragm using a 10 µl Hamilton syringe. Care was taken to prevent the needle from penetrating the thoracic cavity.

Sacrifice--Ten days following each BDA injection and 1 day following each WGA-HRP injection, each animal was deeply anesthetized and transcardially perfused with a mixed aldehyde fixative (1% paraformaldehyde, 1.25% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4). Each brain was removed and stored overnight in cold 10% sucrose buffer. The next day each brain was serially sectioned at 40 µm on a freezing microtome, and the sections were collected into additional cold 0.1 M phosphate buffer. The sections were then histochemically processed to reveal the neuronal labeling which resulted from the various injections: WGA-HRP, BDA, and BDA/ CTb-HRP combination.

In WGA-HRP cases, representative sections were processed with tetramethylbenzidine (TMB) (Mesulam, 1978). A few sections through the medullary injection site were processed with benzidine dihydrochloride (BDHC) (Mesulam, 1976) to establish the center of WGA-HRP uptake.

In cases involving only a BDA injection, the tissues were processed as follows. After thoroughly rinsing in 0.1 M phosphate buffer (pH 7.2), sections were processed for the visualization of BDA label, according to the protocol of Wang and Spencer (1996). Briefly, the sections were preincubated in 1:500 avidin-HRP in 0.1% Triton X-100 phosphate buffer (0. l M, pH 7.2) for at least 4 hours, or overnight in the refrigerator. The sections were thereupon washed with 0.1 M phosphate buffer (pH 7.2), and incubated for 10 minutes with 0.05% diaminobenzidine (DAB) in phosphate buffer (pH 7.2), to which 1% solutions of nickel chloride and cobalt acetate had been added, each at a concentration of 0.5 ml/100 ml. The reaction was initiated, by addition of 3.0% hydrogen peroxide at a concentration of 0.3 ml/100 ml of DAB solution, and lasted for 5­20 minutes. The sections were then washed with phosphate buffer and mounted.

In cases involving both a medullary injection of BDA as well as a diaphragmatic injection of CTb-HRP, the sections had to be processed in a two stage protocol (Smith and Bolam, 1991). In the first step of this process, the sections underwent the modified TMB protocol of Olucha and associates (1985). In this method, the sections were preincubated for 20 minutes in 800 ml of 0.1 M phosphate buffer (pH 6.0) that contained 0.25% ammonium molybdate and 0.005% TMB. Next, 10 ml of 0.3% hydrogen peroxide were added three times at 20 minute intervals for incubation. Incubation took place overnight in the refrigerator and was followed by a stabilization step in a 5.0% ammonium molybdate solution in 0.1 M phosphate buffer, pH 6.0. In the second step of this process, the sections were processed for the BDA reaction as described in the preceding paragraph. This second step, because it involved the use of nickel and cobalt, caused the BDA-containing axons to become black; whereas the first step caused the CTb-HRP-containing somas to become reddish-brown.

Sections from each case were then mounted onto gelatinized slides and counterstained with 1% neutral red. Thereupon, the material was studied under both bright- and darkfield microscopy.

RESULTS

Following the histochemical processing of the tissues, the BDA and WGA-HRP injection sites were readily observed. These injection sites were approximately 300­400 µm in diameter and were located within the CVLM (Fig. 1). Typically, the injection sites were observed at a level immediately caudal to the obex, where they were centered within the nucleus ambiguus, but with some spread into the immediately adjacent reticular formation.

Orthogradely labeled axons were observed to exit each of the injection sites, descend into the spinal cord and terminate within the ventral horns at mid-cervical levels (Fig. 2). Differences in labeling distribution, with respect to cases involving left versus right CVLM injections, were not observed. Furthermore, the labeling patterns observed in BDA and WGA-HRP cases were virtually identical. This labeling pattern will be described in the following paragraph. Axons labeled with either BDA or WGA-HRP were apparent with either bright-field or dark-field microscopy. However, under bright-field microscopy individual axons labeled with BDA were darker, had a thicker appearance, and were consequently more noticeable than axons labeled with WGA-HRP. Furthermore, only BDA labeled axons typically displayed varicosities within the ventral horns. Because these varicosities were not observed within white matter, their presence suggested the existence of synaptic contacts within the ventral horns.

Emerging from a typical injection site, the majority of labeled axons were observed to descend through the ipsilateral reticular formation to enter the lateral and ventral funiculi (Figs. 2D, E; 3A). The majority of these ipsilateral axons were observed within the ventral portion of the lateral funiculus. In addition to the ipsilateral projection, some axons were observed to decussate at various levels of the medulla, prior to their descent through the lateral reticular formation and entry into ventral and lateral funiculi of the contralateral spinal cord (Fig. 2A­C). In general these decussations were diffuse, with some fibers ascending and thereupon crossing through the ventral tegmentum of the rostral medulla, and other fibers crossing through the caudal medulla. Some of these latter fibers decussated immediately dorsal to the central canal, streaming through and around the nucleus of the solitary tract, while other fibers were observed to decussate through the ventral tegmentum of the caudal medulla. In addition to these medullary decussations, some CVLM projections were observed to decussate within the spinal cord (Fig. 2E).

At mid-cervical levels of the spinal cord, many axons were observed to terminate bilaterally within the ventral horns, where spherical terminal fields were noted. The bilaterality of these terminations was most clearly observed in WGA-HRP cases, where the terminal fields were noted to be somewhat similar in size and labeling density (Fig. 3C, D). In these cases, approximately 60­70% of the labeled axons terminated within the contralateral phrenic nucleus, whereas 30­40% of the labeled axons projected to the ipsilateral phrenic nucleus. (In BDA cases, the projection to the ipsilateral ventral horn was largely obscured by the coarse appearance of labeled axons passing through this area [Fig. 3A].) Input to each ventral horn came primarily by way of the labeled axons of the ventral and lateral funiculi, as previously described. In company with the CVLM-phrenic projections, some axons occupying the lateral funiculus were observed to extend caudally, bypassing the phrenic nuclei on their way to more caudal levels of the spinal cord. The terminations of CVLM input to thoracic levels of the spinal cord will be described in a subsequent publication.

In addition to orthogradely labeled axons, neurons retrogradely labeled following the injections (of either BDA or WGA-HRP) into the CVLM were observed within the cervical cord. Neurons retrogradely labeled with WGA-HRP tended to be more numerous and more intensely labeled. Retrogradely labeled neurons were of a moderate size (approx. 15 µm in soma diameter) and multipolar in configuration. Contralateral to the injection, these neurons were primarily located within the ventral horn, often at the periphery of the orthogradely labeled axon terminal field. Ipsilateral to the injection, retrograde labeling tended to be located within the dorsal horn.

Injections of CTb-HRP, made into the diaphragm of three rats, resulted in the retrograde labeling of ipsilateral phrenic motor neurons. Typically, 1­6 neuronal cell bodies were labeled in each section through the mid-cervical cord. In these cases BDA injections had also been made into the CVLM, contralateral to the diaphragm injection, thus labeling the previously described medullospinal projections. As a result of these experiments, input from the CVLM was observed in relation to the phrenic motor neurons (Fig. 4A, B). Specifically, it was noted that BDA-containing (medullospinal) varicosities were located in close proximity to the phrenic motor neurons. These varicosities, distributed as a series of axonal beads (varicosities en passant), often appeared to contact the labeled soma (Fig. 4B).

DISCUSSION

The primary findings of this study were that the CVLM of the rat projects bilaterally (typically with a contralateral predominance) to the phrenic nucleus, whereupon some of the descending axons appear to synapse directly upon phrenic motor neurons. These findings largely confirm the results of previous studies (Ellenberger and Feldman, 1988; Yamada et al., 1988) which suggest that the ventral respiratory group, located largely within the CVLM, communicates with the phrenic motor neurons via direct synaptic contacts. The fact that the projection tended to be heavier to the contralateral phrenic nucleus is supportive of similar findings made in cats (Feldman et al., 1985; Portillo et al., 1986; Holstege, 1989) and rats (Ellenberger and Feldman, 1988). It does not support physiological findings made in cats which suggest that projections of the ventral respiratory group to the phrenic nucleus are exclusively crossed (Merrill, 1970; Hilaire and Monteau, 1976).

To better understand this apparent disparity in findings, it should be noted that the previously cited physiological studies were limited to investigations of respiratory neurons which fired in synchrony with phrenic activity. Conversely, in the present study (as well as the anatomical studies cited above) the focus was upon regional projections to the phrenic nuclei, irrespective of the spontaneous firing patterns of the neurons located within the region. Thus, the projections described in the anatomical studies may reflect a more comprehensive picture of the pathways through which the CVLM influences respiratory functions. At issue is whether or not a neuron must tonically fire in synchrony with phrenic nerve activity in order for it to be considered a "respiratory" neuron. It seems conceivable that some neurons having an influence upon phrenic nerve activity may not be tonically active, and therefore may be quiescent during periods of unlabored respiration (such as those encountered in animal experiments). The labeling of such a population of neurons, having input to the phrenic nuclei, may explain the disparate findings of physiological and anatomical studies.

In this study, labeled axons emanating from the injections sites were observed to decussate diffusely within the rostral and caudal medulla, as well as within the spinal cord at the level of the phrenic nuclei. These observations, regarding the various levels of decussation, tend to be in agreement with similar findings made in the rat (Ellenberger and Feldman, 1988) and the cat (Holstege, 1989). Of considerable interest, it has been noted in rats and cats (Gauthier et al., 1986), but not in monkeys and rabbits (Karczewski and Gromysz, 1982), that a midsagittal section made rostral to the obex is effective in totally blocking phrenic nerve activity. The implications of this finding are that in cats and rats each phrenic nerve requires the input of the contralateral medulla, and that these medullary influences decussate rostral to the obex. The results of such midsagittal sections, as observed in rats (Gauthier et al., 1986), are difficult to resolve with the anatomical findings of the present study. Projections from the CVLM to phrenic nucleus, as shown in the present study, as well as by others (Ellenberger and Feldman, 1988), follow both crossed and uncrossed pathways in rats. Furthermore, only a portion of the axons decussate rostral to the obex; whereas, others decussate at various levels caudal to the obex. Additionally, the uncrossed projection from CVLM to the phrenic nucleus is substantial (Feldman et al., 1985; Yamada et al., 1988). In view of the diversity of these pathways, it is difficult to understand how a single midline lesion made rostral to the obex could abolish phrenic activity. The simplest explanation may be that the CVLM-phrenic decussations occurring in the rostral medulla are the primary drivers of inspiratory (phrenic) function. On the other hand, it may be that rostral medulla midline lesions cause damage to other tracts or regions that are necessary for respiratory drive. Clearly, the functional significance of both the ipsilateral CVLM-phrenic projections, as well as the contralateral CVLM-phrenic fibers which decussate within the caudal medulla and spinal cord need to be further investigated.

It was observed that the great majority of medullospinal projections, emanating from the CVLM, descended ipsilaterally through the lateral and ventral funiculi. However, at their distal termination, the medullospinal axons were slightly more numerous to the contralateral phrenic nucleus. The fact that some CVLM-phrenic axons were observed to decussate within the spinal cord, at the level of phrenic nucleus, partially explains the apparent disparity between the predominance of ipsilateral axon descent and the predominance of contralateral labeling within the phrenic nucleus. Labeled medullospinal axons, however, were also observed within the lateral funiculus at spinal cord levels caudal to the phrenic nucleus. Consequently, it is concluded that some of those axons labeled by injections made into the CVLM were destined for sites located at more caudal levels of the spinal cord. In keeping with this point, it has been reported that within the CVLM are neurons that project to thoracic (Feldman et al., 1985; Holstege, 1989; Blessing, 1990; Matsumoto et al., 1994) and lumbosacral (Holstege and Tan, 1987; Holstege, 1989) levels of the spinal cord. It has been suggested that these projections to thoracic and lumbosacral cord may participate in a variety of functions, including: accessory respiratory functions (Miller et al., 1985; Holstege, 1989), cardiovascular functions (Caverson et al., 1983; Blessing, 1990; Aicher et al., 1992; Hardy and Horecky, 1995), vocalization (Holstege, 1989), sexual posturing (Van der Horst et al., 1994), and micturition (Holstege and Tan, 1987).

The results of the present study suggest that the CVLM projects directly to phrenic motor neurons in rats. This input tends to be slightly greater to the contralateral phrenic nucleus, but there is also a significant input to the ipsilateral phrenic nucleus. Through these pathways the CVLM has the potential to bilaterally influence respiratory functions. Interestingly, the CVLM (in addition to its respiratory functions) also contains neurons that tonically influence cardiovascular functions. Typically, the cardiovascular neurons within the CVLM appear to be involved in vasodepressor mechanisms (Pagani et al., 1983; Imaizumi et al., 1985; Gieroba and Blessing, 1992) although vasopressor responses have also been elicited from this area (Gordon and McCann, 1988; Possas et al., 1994). The close proximity within the CVLM of neurons subserving respiratory and cardiovascular functions tends to suggest that functional interrelationships may exist between these two neuronal populations. As a result, it is conceivable that (existing within the CVLM) a purely neural substrate may exist whereby the cardiovascular and respiratory systems may have the potential to directly influence one another, thus yoking two functions that we know to be intimately related under normal behavioral conditions.

ACKNOWLEDGMENTS

The authors thank Dr. P.J. May and Dr. G.A. Mihailoff for reading this manuscript. We also thank Dr. W. Sun and Dr. P.J. May for technical advice given during the early phases of this project. This study was supported in part by NIH grant 1-R15-NS32861.

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