THE ANATOMICAL RECORD 242:251-258 (1995) Medullary Projections to the Vagus Nerve and Posterolateral Hypot ha1amus S.G. PATRICK HARDY Departments of Physical Therapy and Anatomy, The University of Mississippi Medical Center, Jackson, Mississippi ABSTRACT Background: Vagal visceromotor reflexes are dependent upon reciprocal neural connections existing between the medulla and the hypothalamus. Medullohypothalamic neurons may provide feedback cues to the hypothalamus regarding the activity of vagal motor neurons. As yet, however, studies investigating the spatial relationships between medullohypothalamic neurons and vagal motor neurons have not been performed. Methods: A variety of retrogradely transported tracers were used for the purpose of mapping the relative locations of medullovagal and medullohypothalamic neurons. Tracers were injected into the cervical vagus nerve and/or the posterolateral hypothalamus, and subsequently the retrogradely labeled medullary neurons were plotted. Results: Labeling of the two neuronal populations was primarily observed within the ventrolateral and dorsomedial medulla. Within the ventrolateral medulla, medullovagal neurons were found within the retrofacial nucleus and nucleus retroambiguus, whereas medullohypothalamic neurons were located subjacent to these nuclei. Within the dorsomedial medulla, labeling of the two neuronal populations was primarily limited to the vagal-solitary complex. At this location medullovagal neurons were found within the dorsal vagal nucleus, whereas medullohypothalamic neurons were largely confined to the caudal aspect of the solitary nucleus. Conclusions: Because of the spatial proximity existing between medullovagal and medullohypothalamic neurons, it is suggested that functional interrelationships may exist between these two neuronal populations. Specifically, it is suggested that the medullohypothalamic neurons identified in this study may support vagal-related functions by providing feedback cues to the posterolateral hypothalamus. o 1995 Wiley-Liss, Inc. Key words: Nucleus ambiguus, Vagal-solitary complex, Cardiovascular, Respiratory, Fluorescent tracers, Horseradish peroxidase The vagus nerve consists primarily of the processes from general visceral efferent neurons (preganglionic parasympathetic) and general visceral afferent neurons. These efferent and afferent fibers, each playing a crucial role in the mediation of vagal reflexes, are connected to the medulla. Vagal general visceral efferent fibers originate from the dorsal vagal nucleus and nucleus retroambiguus (the latter nucleus being referred t o by some (Bieger and Hopkins, 1987) as the external formation of the nucleus ambiguus). Axons from these medullary nuclei traverse the vagus nerves and convey visceral motor influences to a wide variety of thoracic and abdominal viscera (Nosaka et al., 1979; Kalia and Mesulam, 1980; Leslie et al., 1982; Bieger and Hopkins, 1987; NunezAbades et al., 1991). Importantly, these vagal motor functions are themselves subject to hypothalamic influences which travel upon diffuse hypothalamomedullary pathways coursing through the lateral tegmentum of the brainstem (Saper et al., 1976; van der Kooy 0 1995 WILEY-LISS. INC. et al., 1984).It has been reported that these descending hypothalamomedullary projections are capable of altering vagal outputs to a variety of viscera, including the stomach (Hara et all, 1990; Namiki et al., 1993; Ma et al., 1994), heart (Cechetto and Chen, 1992), kidneys (Schramm et al., 1993), thyroid/parathyroid glands (Ma et al., 19941, and pancreas (Loewy and Haxhiu, 1993). Vagal general visceral afferent fibers convey information from physiological receptors which are housed within numerous viscera. These receptors include, but are not limited to, stretch receptors in hollow organs and baroreceptors in the aortic arch. The vagal afferents, conducting the viscerosensory information, traverse the vagus nerve and subsequently terminate upon neurons within the nucleus of the solitary tract Received October 17, 1994; accepted January 10,1995. 252 S.G.P. HARDY aseptic conditions. A variety of retrogradely transported tracers (fluorescent and nonfluorescent) were used in the study of medullary neurons having projections to either the posterolateral hypothalamus or vagus nerve. Fluorescent tracers included Diamidinodihydrochloride Yellow (DY) (2% in water), Fast Blue (FBI (2%in water), and Fluorogold (FG) (10%in water). Furthermore, the nonf luorescent tracer, wheat germ agglutinin-horseradish peroxidase (WGA-HRP)(2% in water) was used periodically during this study. A single tracer was used in each of 42 animals, whereas multiple tracers were used in each of 10 animals. It should, furthermore, be noted that three of the tracers used in this study, (i.e., FB, FG, and WGA-HRP)have been reported (Skirboll et al., 1989; Zaborszky and Heimer, 1989)not to be taken up by unbroken axons of passage. Tracer placements made into the posterolateral hypothalamus (n = 30) were accomplished by pressure injection, using either a 5 or 10 p1 Hamilton syringe and a microdrive (David Kopf Instruments, Tujunga, CA) to depress the syringe plunger. To facilitate a localized placement of each of the injectants, a nonbeveled syringe needle was used. Each injection was made over an approximate 10 min period. An additional 5 min was allowed to elapse prior t o removing the needle from the injection site, in order to limit the spread of the injectant. A typical injection volume for the injectants FB, FG, or WGA-HRP was 0.03-0.09 pl, whereas a typical injection volume for the more viscous DY was 0.18-0.27 p1. Tracer placements were made into the vagus nerve (n = 32) using the following method. With the aid of a surgical microscope, the lower cervical portion of the vagus nerve trunk was exposed and gently separated from its connective tissue sheath. The vagus nerve was then severed a t a point immediately superior to the clavicle and the proximal nerve stump inserted into a short (3-5 mm) segment of polyethylene tubing (PE = 901, which had previously been heat sealed on one end and partially filled with one of the tracers used in this study. Leakage typically did not occur during this process. A drop of cyanoacrylate glue was then applied to the tubing entrance, thereby encapsulating the vagal nerve stump and tracer substance. After survival periods of 3-5 days (for the fluorescent tracer cases) and 24 h (for the WGA-HRP tracer cases), the rats were deeply anesthetized with an overdose of chloral hydrate and perfused transcardially with heparinized, normal saline, followed by an aldehyde fixative. For the fluorescent tracer cases, a 2.5% paraformaldehyde fixative was used; however, for the WGA-HRP tracer cases, a 1% paraformaldehyde1.25% glutaraldehyde fixative was employed. The brains and spinal cords were then removed, blocked, immersed in a 10% sucrose-phosphate buffer solution, and refrigerated for several days. Sections were cut on a freezing microtome at 40 pm. Sections taken from WGA-HRP tracer cases were reacted with tetramethMATERIALS AND METHODS ylbenzidine (TMB) (Mesulam, 1978). Tissues taken Experiments were conducted on 52 Sprague-Dawley from fluorescent tracer cases were divided into two adrats weighing 325-450 g. Prior to surgery each animal jacent series. All sections were mounted out of phoswas deeply anesthetized with chloral hydrate (400 mg/ phate buffer onto gelatinized slides. One of the tissue kg). Supplemental doses of the anesthetic were admin- series was left unstained for fluorescence microscopy. istered when the animals demonstrated corneal or with- The other series was counterstained with 1%neutral drawal reflexes. All surgery was performed under red, as were the reacted sections from the WGA-HRP (NTS). Some of the NTS neurons project to neurons in the ventrolateral medulla (Granata et al., 1983; Ross et al., 1985). Many of the neurons within both the NTS (Ricardo and Koh, 1978; Ter Horst et al., 1989; Riche et al., 1990) and ventrolateral medulla (Blessing et al., 1982; Sawchenko and Swanson, 1982) subsequently project their axons upward into the hypothalamus, thus providing a potential source of feedback for hypothalamic influences upon visceromotor functions. Supporting this concept, it has been revealed in physiological studies that medullohypothalamic influences are indeed essential for certain vagal reflexes involving the cardiovascular (Nissen et al., 1993) and gastrointestinal systems (Bray, 1985). Considering the functional interrelationships existing between the vagus nerves and the hypothalamus, it is important to better understand the anatomical bases for those relationships. As yet, however, there are many unanswered questions regarding the neuronal circuitry which would be necessary to support vagalhypothalamic interactions. For example, because most studies involving medullohypothalamic neurons have focused upon those medullary inputs to supraoptic and paraventricular nuclei, relatively little is known regarding medullary input to other hypothalamic areas. Another issue in need of clarification is whether medullohypothalamic and medullovagal neurons originate from discrete neuronal groups or perhaps originate from a common pool of neurons. Data reported in the present paper will shed light on these two areas. The specific medullary outputs examined in the present study were medullovagal neurons, that are channeled through the inferior cervical portion of the vagus nerve, and medullohypothalamic neurons, having projections to the posterolateral hypothalamus. Considerable evidence indicates that the posterolatera1 hypothalamus is largely involved in the regulation of certain cardiovascular functions. For example, it has been observed that activation of the posterolateral hypothalamus produces hypotension and bradycardia (Hinrichsen, 1988; Gelsema et al., 1989; Spencer et al., 1989). Furthermore, it has been demonstrated that the posterolateral hypothalamus mediates emotion-related fluctuations in blood pressure (LeDoux et al., 1988), as well as the cardiovascular effects of prefrontal cortical stimulation (Cechetto et al., 1988; Hardy and Mack, 1990). Interestingly, it has been found that the influence of the posterolateral hypothalamus upon cardiovascular functions is mediated over descending hypothalamomedullary pathways (Cechetto and Chen, 1992). Because the posterolateral hypothalamus is so clearly involved in the regulation of cardiovascular activities, it is concluded that medullary neurons which project to the posterolateral hypothalamus may play a role in cardiovascular functions. A preliminary report of these data has been published elsewhere (Hardy, 1992). 253 MEDULOVAGAL AND MEDULLOHYPOTHALAMIC NEURONS tracer cases. All slides were then coverslipped with D.P.X. mountant. The sections were studied with a Zeiss Photomicroscope 111.An excitation wavelength of 360 nm was used for the purpose of visualizing the fluorescent labeling. The distributions of retrogradely labeled neurons within the medulla were charted with an X-Y plotter (Houston Instruments, Austin, TX) driven by linear potentiometers on the microscope stage. The plots of labeled neurons were transferred to drawings of the adjacent neutral red-stained sections. RESULTS The four retrogradely transported tracers used in this study were found to be highly discriminable because of their different hues and their different cytological labeling properties. Consequently, the distributions of the two populations of medullary neurons focused upon in this study were easily distinguished. Medullohypothalarnic Neurons Medullohypothalamic neurons were labeled following solitary tracer (DY, FB, FG, or WGA-HRP) placements made into the posterolateral hypothalamus. Despite the fact that different tracers were used in this study, the distribution of labeled medullohypothalamic neurons was similar in each case. In every case the tracer placements were made medial to the subthalamic nucleus and lateral to the fornix (see Fig. 1A). Neurons having projections to the posterolateral hypothalamus were observed at a variety of locations within the medulla. In the rostral medulla, moderate numbers of medullohypothalamic neurons were observed ventromedially, within the nucleus raphe magnus, nucleus reticularis magnocellularis, and rostral ventrolateral medulla (Fig. 1B-D). In the caudal medulla, neurons having projections to the posterolateral AP cc CL CM DC F FR G H ICP I0 LRm 2 MTT MV NA NTS OT P PH Rf ST STT SUT VII vs X XI1 A bbreuiations area postrema crus cerebri lateral cuneate nucleus medial cuneate nucleus dorsal cochlear nucleus fornix fasciculus retroflexus gracile nucleus habenula inferior cerebellar peduncle inferior olivary nucleus lateral reticular nucleus, pars magnocellularis lateral reticular nucleus, pars parvocellularis medial lemniscus mammillothalamic tract medial vestibular nucleus nucleus retroambiguus/external formation of the nucleus ambiguus nucleus of the solitary tract optic tract medullary pyramid prepositus hypoglossi nucleus retrofacial nucleus spinal trigeminal nucleus spinal trigeminal tract subthalamic nucleus facial motor nucleus vestibulospinal nucleus dorsal vagal nucleus hypoglossal nucleus hypothalamus were abundant within the ipsilateral nucleus of the solitary tract (Figs. 1F-G, ZB). In addition, numerous medullohypothalamic neurons were observed bilaterallv. within the reticular formation lvinp immediately sugjacent to the nucleus retroambiguus (Figs. 1E,F, ZD, 3). . I " Medullovagal Neurons Neurons retrogradely labeled by tracers applied to the vagus nerve were also observed in both the rostral and caudal medulla. In the rostral medulla, medullovagal neurons were found within and ventral to the ipsilateral retrofacial nucleus (Fig. 1B-D). In the caudal medulla, neurons having projections into the vagus nerve were observed ipsilaterally in both the dorsal vagal nucleus and in the dorsomedial aspect of the nucleus retroambiguus (Figs. 1E-G, ZC, 3). Spatial Relationships Revealed by Multiple Injections By using multiple retrograde tracers, it was possible to observe directly the spatial relationships existing between the two neuronal populations being studied. Data from these experiments served t o confirm the results of the single injectant experiments. Furthermore, the use of multiple injectants made it possible to better recognize those areas in which the two populations of medullary efferents were coextensive. For these purposes, a series of experiments was performed in which two injectants were administered into each animal. Data resulting from these experiments confirmed the locations of medullohypothalamic and medullovagal neurons, as described above. Although double-labeled neurons were never observed, the resulting data also made it possible to observe the spatial interrelationships existing among the two neuronal populations (Figs. 1-3). Furthermore, those areas containing both types of medullary efferents were easily recognized. Examples of distributional overlap (or juxtaposition) were observed in the ventrolateral and dorsomedial medulla. These areas are described below and illustrated in Figure 1. In the ventrolateral medulla, the two populations of neurons were primarily observed in the vicinity of the nucleus retroambiguus (NA) and, to a more limited extent, in the vicinity of the caudal portion of the retrofacial nucleus. In the vicinity of the NA (Figs. ZC, 31, medullovagal projections were observed to originate primarily from the dorsomedial aspect of NA. Medullohypothalamic projections did not originate from the NA but rather from an area immediately ventral to it (Figs. ZD, 3) In the vicinity of the retrofacial nucleus (Fig. 10, medullovagal projections were observed to originate from the retrofacial nucleus, as well as from the subjacent reticular formation. In addition to medullovagal neurons, the subretrofacial region was also observed to contain medullohypothalamic neurons (Fig. lC,D). In the dorsomedial medulla, the two neuronal populations were primarily observed in the caudal portion of the vagal-solitary complex (Figs. 1F,G, ZB). At this location, numerous medullovagal neurons were observed to originate from the dorsal vagal nucleus. Dorsally contiguous with these medullovagal neurons, and residing within the medial portion of the nucleus of the solitary tract, were medullohypothalamic neurons. 254 S.G.P. HARDY CL LRm ICP LRm *-HYPOTHALAMUS 0 -VAGUS HYPO-41 Fig. 1. Illustration of retrogradely labeled medullohypothalamic (stars) and medullovagal (dots) neurons from rostra1 (B) to caudal (G)levels of the medulla following a microinjection (0.27 kl) of Diamidinodihydrochloride Yellow (DY)into the posterolateral hypothalamus (A) and the immersion of the vagus nerve in Fluorogold of a single animal (Hypo-41). DISCUSSION In the present study, the relative locations of medullohypothalamic and medullovagal neurons are de- scribed. The medullohypothalamic neurons that are described in this study are those having projections to the posterolateral hypothalamus, an area well known for MEDULOVAGALANDMEDULLOHYPOTHALAMICNEURONS 255 Fig. 2. A composite of photomicrographs taken of medullovagal and/or medullohypothalamic neurons located within the caudal portion of the vagal-solitary complex (A,B) and within the vicinity of the nucleus retroambiguus (C,D). A Brightfield photomicrograph of the caudal aspect of the dorsomedial medulla. The tissue has been stained with neutral red in order to reveal the regional cytoarchitecture. The rectangle encompasses most of the nucleus of the solitary tract (NTS) and the dorsal motor nucleus of the vagus nerve (XI. B: Fluorescent photomicrograph of retrogradely labeled neurons, corresponding in location to the rectangle of A but photographed in an adjacent, unstained section. A t the top of the photomicrograph, located within the nucleus of the solitary tract, are Fluorogold-labeled medullohypothalamic neurons. At the bottom of the photomicrograph, located within the dorsal motor nucleus of the vagus nerve, are medullovagal neurons which have been labeled following the immersion of the vagus nerve in Diamidino-dihydrochlorideYellow. C: Fluorescent photomicrograph of Fluorogold-labeled medullovagal neurons. The tissue has been stained with neutral red, thus making it possible to observe that the medullovagal neurons are located in the dorsal aspect of the nucleus retroambiguus (NA). D Brightfield photomicrograph of WGAHRP-labeled medullohypothalamic neurons. Note that the labeled neurons are subjacent to the nucleus retroambiguus (NA). its involvement in cardiovascular activities (Cechetto et al., 1988; Hinrichsen, 1988; LeDoux et al., 1988; Gelsema et al., 1989; Spencer et al., 1989; Hardy and Mack, 1990). Consequently, the medullohypothalamic neurons which are identified in this study are likely to play a role in cardiovascular functions. Conversely, it is somewhat more difficult to link the medullovagal neurons, retrogradely labeled in this study, with a particular function. This is largely due to the fact that the vagus nerve is involved in a variety of functions, most of which are dedicated to the control of one’s internal environment. Medullohypothalamic neurons, as observed in this study, were distributed in a manner which varied only slightly from previous reports (Ricardo and Koh, 1978; Blessing et al., 1982; Sawchenko and Swanson, 1982; Ter Horst et al., 1989;Riche et al., 1990). Interestingly, none of these previous studies were designed to comprehensively identify medullohypothalamic inputs which were specific to the posterolateral portion of the hypothalamus. Instead, some of these studies were intended to identify various medullary neurons which projected to the paraventricular (Blessing et al., 1982; Sawchenko and Swanson, 1982) and supraoptic nuclei (Sawchenko and Swanson, 1982), while other studies were designed to reveal projections from the nucleus of the solitary tract (NTS) to the hypothalamus in general (Ricardo and Koh, 1978; Ter Horst et al., 1989; Riche et al., 1990). Nevertheless, the findings of these earlier studies tend to be in agreement with the findings of the present study. For example, previous studies indicated that NTS-hypothalamic projections originated from the caudal and medial portions of the NTS (Ricardo and Koh, 1978; Ter Horst et al., 1989; Riche et al., 1990) and that some of these projections terminated within the posterolateral portion of the hypothalamus (Ricardo and Koh, 1978).Both of these observations are consistent with the findings of the present study. 256 S.G.P. HARDY Fig. 3. Fluorescent photomicrographs of retrogradely labeled neurons within and ventral to the nucleus retroambiguus (NA). A The perimeter of the NA is indicated by the dotted line. Medial is to the right, and dorsal is at the top. Fluorogold-labeled medullovagal neurons (arrows) are primarily located in the dorsomedial NA. Subjacent to the NA are medullohypothalamic neurons (closed arrowheads) which were retrogradely labeled following a microinjection of Diami- dino-dihydrochloride Yellow made into the posterolateral hypothalamus. (Open arrowheads indicate the presence of medullospinal neurons within the ventral NA. These medullospinal neurons are the focus of another study, and the data regarding these neurons have been excluded from the present study.) B Higher magnification of the medullohypothalamic neurons, as seen within the square of A. Closed arrowheads, medullohypothalamic neurons. It has been demonstrated (Sawchenko and Swanson, 1982; Blessing et al., 1982) that the paraventricular nucleus receives input from the NTS and the ventrolateral medulla. The fact that neurons in these areas also project to the posterolateral hypothalamus, as demonstrated in the present study, suggests that these neurons may be able to concomitantly influence both the paraventricular nucleus and the posterolateral hypothalamus. Interestingly, it has been well documented that each of these hypothalamic sites plays important roles in cardiovascular regulation. The similarity of medullohypothalamic inputs into these two hypothalamic sites may contribute to their functional similarities. Conversely, in previous studies (Blessing et al., 1982; Sawchenko and Swanson, 1982), neurons in the ventromedial and paraolivary regions were not observed to project to the paraventricular nucleus, whereas in the present study a modest number of neu- rons, originating from these regions of the rostra1 medulla, were observed to project to the posterolateral hypothalamus (Fig. 1B-D). Thus medullohypothalamic inputs originating from the ventromedial and paraolivary regions may be more exclusively related to the posterolateral hypothalamus than those originating from the ventrolateral medulla and nucleus of the solitary tract. Medullovagal neurons, as observed in this study, were distributed in a manner similar to that described in earlier reports (Leslie et al., 1982; Bieger and Hopkins, 1987). Accordingly, medullovagal neurons were typically observed within: the dorsal vagal nucleus, the retrofacial nucleus, and the nucleus retroambiguus. Previous studies (Nosaka et al., 1979; Pagani et al., 1983; Imaizumi et al., 1985; Bieger and Hopkins, 1987) have indicated that vagal cardioinhibitory neurons are primarily located within the dorsal vagal nucleus and MEDULOVAGALANDMEDULLOHYPOTHALAMICNEURONS within the nucleus retroambiguus. Conversely, medullovagal neurons located within the retrofacial nucleus primarily innervate the esophagus (Bieger and Hopkins, 1987). From the multiple tracer experiments, it was observed that medullohypothalamic and medullovagal neurons were in close proximity within the ventrolatera1 and dorsomedial regions of the caudal medulla, where they resided within the caudal portion of the vagal-solitary complex and within the vicinity of the nucleus retroambiguus. Interestingly, and as previously noted, these areas are known to contain cardioinhibitory neurons (Nosaka et al., 1979; Pagani et al., 1983;Imaizumi et al., 1985; Bieger and Hopkins, 1987). In the caudal portion of the vagal-solitary complex, medullohypothalamic neurons were observed within the nucleus of the solitary tract, whereas medullovagal neurons occupied an immediately subjacent location, within the dorsal vagal nucleus. In the caudal portion of the ventrolateral medulla, medullovagal neurons were present within the nucleus retroambiguus. Medullovagal neurons were typically located in the dorsomedial aspect of this nucleus. This observation is in agreement with the findings of previous studies (Nunez-Abades et al., 1991, 1992). Medullohypothalamic neurons were located immediately subjacent to the nucleus retroambiguus but were not observed within the nucleus itself. This latter finding is consistent with the previous observations of others (Blessing et al., 1982; Sawchenko and Swanson, 1982). As revealed in this study, it seems apparent that the caudal portions of the dorsomedial and ventrolateral medulla, because of their neuronal content, have the potential to influence those functions associated with the vagus nerve and the posterolateral hypothalamus. It is suggested that some of the medullohypothalamic neurons identified in this study, and particularly those lying in close proximity to medullovagal neurons, may provide feedback information to the hypothalamus regarding the extent to which the hypothalamus has influenced the activity of medullovagal neurons. Such a system would be consistent with the viewpoint that “corollary discharge” (Sperry, 1950), also known as “efference copy” (Holst and Mittelstaedt, 19501, is an organizational feature existing within all motor systems, providing feedback to higher levels of the neuraxis regarding the motor output occurring at lower levels. In support of this concept as it relates to vagal motor functions, it has been demonstrated that medullohypothalamic neurons are essential for the occurrence of certain vagal motor reflexes (Bray, 1985; Nissen et al., 1993). It should also be noted that most of the medullohypothalamic neurons were located either within the ventrolateral medulla or within the nucleus of the solitary tract. The fact that both of these areas receives vagal afferent input (Granata et al., 1983; Ross et al., 1985) provides additional support for the notion that these medullohypothalamic neurons may provide feedback to the hypothalamus. The anatomical data gathered in the present study demonstrate the spatial interrelationships among medullovagal and medullohypothalamic neurons. It is anticipated that future studies will be directed toward better understanding any functional interrelationships existing among these neurons. 257 ACKNOWLEDGMENTS The author thanks S.M. 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