Brain stem projections of sensory and motor components of the vagus complex in the cat: I. The cervical vagus and nodose ganglion

The motor and sensory connections of the cervical vagus nerve and of its inferior ganglion (nodose ganglion) have been traced in the medulla oblongata of 32 adult cats with the neuroanatomical methods of horseradish peroxidase (HRP) histochemistry and amino acid autoradiography (ARG). In 14 of these subjects, an aqueous solution of HRP was applied unilaterally to the central end of the severed cervical vagus nerve. In 13 other cases, HRP was injected directly into the nodose ganglion. Three of these 13 subjects had undergone infranodose vagotomy 6 weeks prior to the HRP injection. A mixture of tritiated amino acid was injected into the nodose ganglion in five additional cats. The retrograde transport of HRP yielded reaction product in nerve fibers and perikarya of parasympathetic and somatic motoneurons in the medulla oblongata. Furthermore, a tetramethyl benzidine (TMB) method for visualizing HRP enabled the demonstration of anterograde and transganglionic transport, so that central sensory connections of the nodose ganglion and of the vagus nerve could also be traced. The central distribution of silver grain following injections of tritiated amino acids in the nodose ganglion corresponded closely with the distribution of sensory projections demonstrated with HRP, thus confirming the validity of HRP histochemistry as a method for tracing these projections. The histochemical and autoradiographic experiments showed that the vagus nerve enters the medulla from its lateral aspect in multiple fascicles and that it contains three major components—axons of preganglionic parasympathetic neurones, axons of skeletal motoneurons, and central processes of the sensory neurons in the nodose ganglion. Retrogradely labeled neurons were seen in the dorsal motor nucleus of X(dmnX), the nucleus ambiguus (nA), the nucleus retroambigualis (nRA), the nucleus dorsomedialis (ndm) and the spinal nucleus of the accessory nerve (nspA). The axons arising from motoneurons in the nA did not traverse the medulla directly laterally; rather, all of these axons were initially directed dorsomedially toward the dmnX, where they formed a hairpin loop and then accompanied the axons of dmnX neurons to their points of exit. Afferent fibers in the vagus nerve reached most of the subnuclei of the nTS bilaterally, with the more intense labeling being found on the ipsilateral side. Labeling of sensory vagal projections was also found in the area postrema of both sides and around neurons of the dmnX. These direct sensory projections terminating within the dmnX may provide an anatomical substrate for vagally mediated monosynpatic reflexes. Following deefferentiation by infranodose vagotomy 6 weeks prior to HRP injections into the nodose ganglion, a number of neurons in the dmnX were still intensely labeled with the HRP reaction product. The axons of these HRP-labeled perikarya may constitute the bulbar component of the accessory nerve.     

Brainstem projections of sensory and motor components of the vagus nerve in the rat

The sensory and motor connections of the cervical vagus nerves and of its inferior ganglion (nodose ganglion) have been traced in the medulla and upper cervical spinal cord of 16 male Wistar rats by using horseradish peroxidase (HRP) neurohistochemistry. The use of tetramethyl benzidine (TMB) as the substrate for HRP permitted the visualization of transganglionic and retrograde transport in sensory nerve terminals and perikarya, respectively. The vagus nerve in the rat enters the medulla in numerous fascicles with points of entry covering the entire lateral aspect of the medulla extending from level +4 to - 6 mm rostrocaudal to the obex. Fascicles of vagal sensory fibers enter the dorsolateral aspect of the medulla and travel to the tractus solitarius (TS) which was labeled for over 8.8 mm in the medulla. The caudal extent of the TS receiving vagal projections was found in lamina V of the cervical spinal cord (C1 to C2). Sensory terminal fields could be visualized bilaterally in the nucleus of the tractus solitarius (nTS), area postrema (ap) and dorsal motor nucleus of the vagus nerve (dmnX). The ipsilateral projection to the nTS and the dmnX was heavier than that found on the contralateral side. The area postrema was intensely labeled on both sides. Motor fibers from HRP-labeled perikarya in the dmnX travel ventromedially in a distinct fascicle and subsequently subdivide into a number of small fiber bundles that traverse the medullary reticular formation in the form of a fine network of HRP-labeled fibers. As these fibers from the dmnX approach the ventrolateral aspect of the medulla they are joined by axons from the nucleus ambiguus (nA), nucleus retroambigualis (nRA) and the retro facial nucleus (nRF). These latter fibers form hairpin loops in the middle of the reticular formation to accompany the axons from the dmnX exiting from the medulla in a ventrolateral location. HRP-labeled perikarya, in contrast to transganglionically transported HRP in sensory terminals in the nTS, were visualized on one side only, thus indicating that motor control via the vagus nerve is exerted only by motor neurons located ipsilaterally. Sensory information on the other hand, diverges to many nuclear subgroups located on both sides of the medulla.     

Brainstem cells of origin of the cervical vagus and cardiopulmonary nerves in the neonatal pig (Sus scrofa)

The distribution of the cells of origin of the cervical vagus and cardiopulmonary nerves has been studied in neonatal piglets (Sus scrofa) ranging in age from 1 to 60 days. Cardiopulmonary nerves were identified physiologically and anatomically prior to injection of horseradish peroxidase (HRP) into the nerves. Following injection of HRP into the cervical vagus nerve retrogradely labeled neurons were present in the dorsal motor nucleus of the vagus nerve (DMV), the nucleus of the solitary tract, the nucleus ambiguus (NA), ventrolateral to the NA and in an intermediate zone between the DMV and the NA. Two unique clusters of neurons were also retrogradely labeled after injections into the vagus nerve. One group was located lateral to the most caudal levels of the DMV and extended as far caudally as the C1 spinal segment. The second distinctive group was located ventrolateral to the nucleus ambiguus in a cell column identified as the ventrolateral nucleus ambiguus (VLNA). After injections of HRP into cardiopulmonary nerves, the majority of neurons were found in the VLNA and the distinct clusters of neurons in this cell column were particularly heavily labeled. Small numbers of cells were labeled in the DMV and NA and none were labeled in the solitary nucleus after cardiopulmonary nerve injections. There were no apparent age-related differences in the degree or distribution of retrograde labeling.The distribution of neurons in the medulla oblongata projecting into cardiopulmonary nerves in the piglet is similar to that described in other species, i.e., the nucleus ambiguus, particularly its ventrolateral cell column, is the primary site of cardiomotor neurons. In addition, in the piglet there is a morphologically distinct cluster of cells related to the heart, and possibly the lungs, which does not appear to be present in other species.     

Brain stem localization of vagal preganglionic neurons

The central distribution of vagal preganglionic neurons has been examined using the retrograde transport of horseradish peroxidase (HRP). In 27 adult cats, the entire vagus nerve was exposed to HRP. In 13 other cats we examined the brain stem following microinjections of HRP (10 μ1) into individual visceral organs — lung, heart and stomach. Comparison of individual cases led to the conclusion that different patterns exist for each visceral organ. The preganglionic (parasympathetic) innervation of the entire vagus nerve arises from the dorsal motor nucleus of the vagus (dmnX), nucleus ambiguus (nA), nucleus retroambigualis (nRA), nucleus dorso-medialis (ndm), spinal nucleus of the accessory (nspA) and from the reticular formation between the dmnX and nA. Axons arising from the nA do not traverse the medulla laterally; rather they are initially directed dorso-medially toward the dmnX where they bend at right angles and accompany axons of neurons in the dmnX. The motor nuclei innervating the lungs, heart and stomach are the dmnX, the nA and nRA: the dmnX contributes fibers to the heart, lungs and stomach from a region of 10 mm of medulla rostrocaudally; the nA contributes efferents to the 3 viscera studied from the entire 6 mm contributing vagal efferents; the nRA contributes efferents to the stomach in addition to providing innervation to the larynx and trachea (see 19).The area postrema (ap) receives afferent input from the lungs, heart and stomach, as indicated by extraperikaryal grains of HRP reaction product resulting from transganglionically transported HRP (through the ganglion nodosum). Sensory terminal labeling in the various subnuclei of the nucleus of the tractus solitarius (nTS) was also examined and it was found that no specific region of the medulla is devoted to receiving input from any one visceral organ; rather the rostro-caudal extent of vagal afferent terminals in the medulla spans the entire length of the medulla. Differences between the central representation of different viscera seemed to lie within the organization of the nuclear subgroups of the nTS.     

Viscerotopic representation of preganglionic efferent vagus nerve in the brainstem of the rat: a Fluoro-Gold study.

To investigate the viscerotopic distribution of the cells of origin of preganglionic vagus nerve in rats, Fluoro-Gold was injected into various visceral tissues. After injections into the gastroesophageal junction and the gastric corpus, labelled cells were localized in the medial half of the dorsal motor nucleus of the vagus (dmnX). Cells in the nucleus ambiguous (nA) were also labelled after injections into the gastroesophageal junction. After injections into the pancreatic head and the celiac plexus, labelled cells were located bilaterally in the lateral part of the caudal dmnX. In the rostral dmnX, however, the pancreatic head was represented in the medial segment. After injections into the lung, duodenum, liver and ascending colon, no labelling was observed in the brainstem.     

Vagal and gastric connections to the central nervous system determined by the transport of horseradish peroxidase

Horseradish peroxidase (HRP, Sigma Type VI) crystals were encased in a parafilm envelope and applied to the transected central ends of the left and right cervical vagus nerves and the anterior and posterior esophageal vagus nerves of adult male hooded rats. Injections of 30% HRP were made into the muscle wall of the fundus and antrum regions of the stomach. After 48 hr survival time, animals were perfused intracardially with a phosphate buffer plus sucrose wash followed by glutaraldehyde and paraformaldehyde fixative. The brain stem, spinal cord and corresponding dorsal root ganglia, superior cervical sympathetic ganglion, and the nodose ganglion were removed and cut into 50 micron sections. All tissue was processed with tetramethylbenzidine (TMB) for the blue reaction according to Mesulum and counterstained with neutral red. Sequential sections were examined under a microscope. Labeled neurons and nerve terminals were identified using bright and dark field condensers and polarized light. In tissue from animals that had HRP applied to the cervical vagus nerves, retrogradely labeled neurons were identified ipsilaterally in the medulla located in the dorsal motor nucleus of the vagus (DMN) and the nucleus ambiguus (NA). Labeled cells extended from the DMN into the spinal cord in ventral-medial and laminae X regions C1 and C2 of cervical segments. Many neurons were labeled in the nodose ganglion. Anterogradely labeled terminals were observed throughout and adjacent to the solitary nucleus (NTS) dorsal to the DMN and intermixed among labeled neurons located in the DMN. In tissue from animals that had HRP applied to the esophageal vagus nerves, similar labeling was observed. However, fewer neurons were identified in the NA, the nodose ganglion, and only in laminae X of the cervical spinal cord segments C1 and C2. Also, very little terminal labeling was observed in and adjacent to the NTS. Labeled neurons in tissue from animals that had HRP injected into the stomach wall were observed bilaterally in the DMN, nodose ganglion, and only in laminae X at the C1 and C2 levels of the spinal cord. Labeled neurons also were observed in the dorsal root ganglia of the thoracic cord. These data indicate that cervical cord and NA neurons are important in the supradiaphragmatic motor innervation by the vagus. Also, many afferents to the NTS originate above the diaphragm. In addition, some afferents from the stomach enter the central nervous system via the thoracic spinal cord.     

Brainstem cells of origin of physiologically identified cardiopulmonary nerves in the rhesus monkey (Macaca mulatta)

The distributions of brainstem cells of origin of the cervical vagus nerve, its cervical and thoracic branches, and of neurons projecting to the cricothyroid muscle and the stomach wall were identified and compared following injections of horseradish peroxidase (HRP) in 18 Rhesus monkeys. Physiologically and/or anatomically identified cardiopulmonary nerves were injected with 3–20 μl of HRP to identify the locations of vagal preganglionic cardioinhibitory neurons in 10 of these monkeys. After injections into cardiopulmonary nerves, retrogradely labelled cells were concentrated ipsilaterally in the most lateral parts of the dorsal motor nucleus of the vagus nerve (DMV) and in the ventrolateral nucleus ambiguus (NA). Fewer labelled neurons were identified close to or in the principal (dorsal) division of the NA and in the intermediate zone between the DMV and NA. The results indicate that monkey cardiopulmonary nerves have multiple origins; their somata are located primarily in the ventrolateral NA and to a lesser extent in the lateral DMV. In monkeys, there is a stronger representation in the lateral DMV than in cat, dog and pig. The viscerotopic organization of the cells of origin of primate vagal nerves is similar to that in other species. The cells of origin of vagal projections to the superior laryngeal nerve and cricothyroid muscle were located in the NA rostrally to those of the inferior laryngeal nerve. Injections into the superior laryngeal nerve also resulted in significant labelling in the DMV and intermediate zone (IZ). The cells of origin of projections to the anterior stomach wall were restricted to the DMV with a bilateral distribution of labelled cells, concentrated medially in the nucleus.     

The central distribution of the cervical vagus nerve and gastric afferent and efferent projections in the rat

The medullary distribution of afferent fibers and cells of origin of the cervical vagal trunk and of the vagal innervation of the stomach have been studied using the anterograde and retrograde transport of horseradish peroxidase (HRP). Injections of HRP were made into the cervical vagus nerve, the stomach wall, the proximal small intestine, or the peritoneal cavity. Two to four days following the injections, the rats were perfused and the medullae oblongatae and nodose ganglia were processed using the tetramethyl benzidine method. Cervical vagus nerve injections of HRP resulted in heavy anterograde labeling in the ipsilateral nucleus of the tractus solitarius (NTS) and the commissural nucleus. Lighter labeling was seen in these regions on the contralateral side, but did not extend as far rostrally in the NTS. Labeling was also seen in the area postrema. Retrogade labeling of somata was present in the ipsilateral side in the nodose ganglion, throughout the whole extent of the dorsal motor nucleus of the vagus, much of the nucleus ambiguus and in rostral levels of the cervical spinal cord. After stomach injections, labeling indicative of afferent fibers was observed bilaterally in the dorsomedial and medial portions of the NTS and in the commissural nucleus. Labeled efferent fibres arose from neurons in the dorsal motor nucleus of the vagus, nucleus ambiguus and the cervical spinal cord. Retrogradely labeled somata were found bilaterally, throughout the rostrocaudal length of the dorsal motor nucleus in all cases with stomach injections. In some, but not all cases, labeled somata were seen bilaterally in compact areas within the nucleus ambiguus, particularly rostrally. Control injections of HRP into the intestinal wall and peritoneal cavity indicated that the stomach was the primary source of afferent and efferent labeling in the medulla following subdiaphragmatic injections.     

Morphology of identified preganglionic neurons in the dorsal motor nucleus of the vagus.

To determine the degree of variation of neuronal morphology both within and between the subnuclei of the dorsal motor nucleus of the vagus (dmnX), structural features of the preganglionic neurons of each of the five primary subnuclei in the rat dmnX were characterized quantitatively. Each of the columnar subnuclei was separately labeled by application of the retrograde tracer fast blue to its corresponding subdiaphragmatic vagal branch. Fixed brain slices of 100 microns thickness were then prepared in coronal, sagittal, and horizontal orientations. Next, randomly selected fast blue labeled neurons (n = 1,256) were injected with Lucifer yellow, drawn with camera lucida, and digitized. For each cell, three features of the perikaryon and twelve of the dendritic tree were measured. Dorsal motor nucleus neurons with up to eight primary dendrites, 30 dendritic segments, and seventh order dendritic branches were observed. Throughout the dmnX, the dendrites of preganglionic neurons were preferentially oriented in the horizontal plane. Consistent with an organizing role for the columnar subnuclei, most dendrites remained within their column of origin. However, between 5 and 30% of the neurons in each of the columns projected dendrites into adjacent dmnX subnuclei or other brainstem nuclei, including the nucleus of the solitary tract (NTS). The cyto- and dendroarchitectural analyses revealed systematic gradations in morphology, although they did not support the idea that the dmnX was composed of multiple distinct preganglionic types. The most parsimonious interpretation of the data is that dmnX motorneurons are variants of a single prototype, with dendrites varying widely in length and degree of ramification. The extent of an individual preganglionic neuron's dendritic field was predicted by three factors: the cell's rostrocaudal position within the dmnX, its location within a transverse plane (i.e., its coronal position within or ectopic to the dmnX), and its subnucleus of origin. Neurons at rostral and midlongitudinal levels of each column had more extensive dendritic arbors than those at caudal levels. Ectopic neurons had more extensive dendritic fields than similar cells in the corresponding columns; in fact, of all vagal preganglionic neurons, ectopics had the most extensive dendritic fields. Somata and dendrites of celiac column neurons were more extensive than those of hepatic and gastric column cells. These differential regional distributions of vagal preganglionics suggest that their structure and function are correlated.     

Contribution of the vagus nerve to angiotensin II binding sites in the canine medulla

Specific angiotensin II (Ang II) binding sites are present in the dorsal medulla of several species and dose-related cardiovascular effects are produced by microinjection of the peptide into this region. Because the anatomical location of Ang II binding sites in the area postrema (ap), nucleus tractus solitarii (nTS) and dorsal motor nucleus of the vagus (dmnX) coincides with the topography of vagal afferent fibers and efferent motor neurons, the effect of either nodose ganglionectomy or cervical vagotomy on Ang II binding sites in the dorsomedial medulla was investigated in dogs by in vitro receptor autoradiography. Two weeks after unilateral ganglionectomy, there was a marked reduction in the density of specific Ang II binding sites in the ipsilateral ap, nTS and dmnX and an absence of binding sites in the region where vagal afferent fibers course through the rostral medulla. Unilateral cervical vagotomy, which has been shown to spare central processes of afferent fibers, resulted in a loss of binding only in the ipsilateral dmnX. We also show that Ang II binding sites are present in the nodose ganglion and central and peripheral processes of the vagus nerve. The data indicate that medullary Ang II binding sites are associated with both vagal afferent fibers and efferent motor neurons.     

The origin and development of the vagal and spinal innervation of the external muscle of the mouse esophagus

Retrograde and anterograde tracing and immunohistochemical techniques were used to examine the origin of the extrinsic innervation, and the development of the vagal innervation to the mouse esophagus. Cholinergic nerve terminals were localised using an antiserum to the vesicular acetylcholine transporter and cholinergic cell bodies were localised using an antiserum to choline acetyltransferase. Cholinergic nerve terminals, which also contained calcitonin gene-related peptide, were present at the motor end plates in the external (striated) muscle of the esophagus. Following injection of Fast Blue into subdiaphragmatic or cervical levels of the esophagus, the only retrogradely-labelled cholinergic nerve cell bodies that also contained calcitonin gene-related peptide were found in the nucleus ambiguus. Neurons in the dorsal motor nucleus of the vagus, the nodose ganglia and dorsal root ganglia gave rise to a number of different types of nerve terminals within the myenteric plexus. Retrogradely-labelled neurons in the dorsal motor nucleus of vagus contained cholinergic markers only, nitric oxide synthase only or cholinergic markers plus nitric oxide synthase, retrogradely-labelled neurons in the dorsal root ganglia contained calcitonin gene-related peptide only, and a small number of retrogradely-labelled neurons in the nodose ganglia contained tyrosine hydroxylase. The development of the vagal innervation to the esophagus was examined following application of DiI to the vagus nerve of fixed mouse embryos. Anterogradely-labelled nerve fibres, which arose from both nodose ganglia and the medulla, were already present in the esophagus of embryonic day 12 (E12) mice. Some of the DiI-labelled vagal nerve fibres were present in among the smooth muscle cells of the external muscle layer prior to their transdifferentiation to striated muscle. We conclude that the neurons in the nucleus ambiguus that project to the esophagus differ from other extrinsic neurons in their chemistry as well as their targets within the esophagus. The development of the extrinsic innervation precedes the transdifferentiation of the external muscle to striated muscle, raising the possibility that, during development, smooth muscle of the esophagus is innervated transiently by vagal neurons.     

Localization and acetylcholinesterase content of vagal efferent neurons

The acetylcholinesterase (AChE) content of rat vagal efferent neurons was studied. Retrograde transport of horseradish peroxidase (HRP) by cut vagal axons provided a means for localizing efferent cell bodies; tissue sections were then processed for the simultaneous visualization of HRP and AChE. A dorsal vagal efferent column contained the dorsal motor nucleus of the vagus, as a primary component, and extended caudally into the upper cervical spinal cord. A ventral column contained neurons in the nucleus ambiguus and the surrounding reticular formation. Although most of the vagal efferent neurons stained with moderate to heave intensity for AChE there were some HRP-labeled cells that contained little AChE and a small percentage in which AChE was absent. In spite of the fact that AChE has been demonstrated in certain non-cholinergic neurons, it has also been found in all cholinergic neurons. Therefore, the presence of AChE has been regarded as a necessary (but not sufficient) component for identifying cholinergic neurons. The absence of AChE in a small percentage of the vagal efferent neurons indicates that some preganglionic parasympathetic fibers in the vagus nerve are not cholinergic.     

Identification and localization of the motor nuclei and sensory projections of the glossopharyngeal, vagus, and hypoglossal nerves of the cockatoo (Cacatua roseicapilla), Cacatuidae

Horseradish peroxidase (HRP) has been applied to the proximal severed ends of glossopharyngeal (N IX), vagus (NX), and hypoglossal (N XII) cockatoo in order to localize the motoneurons and sensory projections of these nerves which are involved in the control of the bird's feeding and phonatory behaviors. Application of HRP to N IX labeled four rhombencephalic nuclei: (1) a large-celled, retrofacial nucleus supplying M. geniohyoideus, the major tongue extensor; (2) a dorsal nucleus composed of medium-sized cells, projecting to most branches of N IX; (3) a ventrolateral nucleus supplying, amongst other structures, the floor of the pharynx and larynx; and (4) a ventral portion of the dorsal motor nucleus of the vagus. Neurons labeled by application of HRP to the cervical vagus comprise the classically defined dorsal motor nucleus and a ventrolateral medullary nucleus which is coextensive with that of the glossopharyngeus: together they probably constitute a nucleus ambiguus. Application of HRP to hypoglossal branches labeled a large nucleus intermedius (IM) and neurons ventral, ventrolateral, and caudal to it. The rostral third of IM supplies the lingual muscles, the caudal two-thirds the tracheosyringeal muscles. Many labeled neurons were found in the "jugular" ganglion following HRP treatment of each of the three nerves, especially N IX and N XII, which innervate the tongue. Central projections of these neurons are to nuclei of the descending trigeminus and to largely nonoverlapping portions of the principal trigeminal nucleus. It is hypothesized that these afferents provide sensory information necessary for the efficient processing and passage of food in the mouth.     

Retrograde axonal transport of horseradish peroxidase in peripheral autonomic nerves.

An exogeneous marker protein, horseradish peroxidase (HRP) was used to race peripheral autonomic pathways in adult guinea pigs and cats. Small doses of HRP were injected into various organs and after a brief survival period, HRP activity appeared in the perikarya of autonomic neurons that supplied each injection site. After injection of HRP into the anterior chamber of the eye, reaction product was detected in the postganglionic sympathetic neurons of the superior cervical sympathetic ganglion. In another experiment, HRP reaction product was found in the cell bodies of the preganglionic sympathetic neurons that supply the adrenal medulla. These were located in the lateral gray column of the spinal cord at T6 and T7 segmental levels. Reaction product appeared in intramural postganglionic parasympathetic neurons close to an injection site in the wall of the urinary bladder and in a similiar situation in Meissner's ganglia of the ileum. Following injection into the walls of the stomach and ileum, HRP labelled cells were detected in the nodose ganglion of the vagus and in preganglionic parasympathetic neurons in the dorsal motor nucleus of this nerve. After injection into the subepicardial tissue of the heart, reaction product appeared in the stellate ganglion and also in an upper thoracic dorsal root ganglion. These data suggest that HRP is taken up by peripheral autonomic nerves of all types, and then undergoes rapid retrograde axonal transport to the perikaryon. It appears, therefore, that HRP may be useful in tracing both motor and sensory peripheral autonomic pathways.     

Connections of a vagal communicating branch in the ferret I. Pathways and cell body location

In contrast to most other species, ferrets possess a single communicating branch connecting the dorsal and ventral vagal trunks immediately rostral to the diaphragm. This branch is being used in physiological studies of gastrointestinal function and emesis. However, the fibre routes which pass through this branch are not known. In this study, the afferent and efferent pathways within this supradiaphragmatic vagal communicating branch of the ferret were studied through the use of the horseradish peroxidase (HRP) tracing technique. The region of the branch was exposed using a thoracotomy and HRP crystals were applied to one of the following: (A) the ventral end of the communicating branch, (B) the dorsal end of the communicating branch, (C) the distal end of the dorsal vagal trunk rostral to the communicating branch or (D) the distal end of the ventral vagal trunk rostral to the communicating branch. Following a 72 hour survival period, the animals were reanaesthetized and perfused. The superior cervical and nodose ganglia and the brain stem were processed using the tetramethylbenzidine method. Following application of HRP to the cut ventral end of the communicating branch, labelled cell bodies were found in the left and right nodose ganglia and in the left dorsal motor nucleus of the vagus. After HRP application to the cut dorsal end of the communicating branch, labelled cells were found in the left and right nodose ganglia. No HRP containing cell bodies were found following HRP application to the cut distal end of either the dorsal or the ventral vagal trunk. These results indicate that several afferent pathways exist within the branch, although only one consistently labelled efferent pathway was found.     

The target specificity of the extrinsic innervation of the rat small intestine

The target specificity of the extrinsic innervation of the rat small intestine was examined by simultaneously injecting the proximal and distal small intestine with either wheat germ agglutinin-horseradish peroxidase (HRP) or fast blue. The number of single- and double-labeled cells in the nodose, dorsal root and coeliac-superior mesenteric ganglia and the dorsal motor nucleus of the vagus were counted and expressed as percentages of total labeled cells. Cells containing both HRP and Fast blue projected to both regions of the intestine. We found that the nodose and mesenteric ganglia contained significantly fewer double-labeled neurons (approximately 3 and 9% respectively) than the dorsal motor nucleus (19%) or dorsal root ganglion (20%). Presumably, a large number of double-labeled afferent or efferent neurons would limit the ability of a given component of the extrinsic innervation to control the activity of restricted regions of the small intestine (but might be important in overall regulation of intestinal function). In a separate series of experiments we examined the topography of neurons in the dorsal motor nucleus of the vagus labeled with HRP injection into either the proximal or distal small intestine. Both of these injections labeled neurons in the entire rostro-caudal extent of the nucleus, though approximately 75% of the cells were located between 720 microns caudal and 720 microns rostral to the obex. Cells in the rostral regions were found primarily in the lateral pole of the nucleus, whereas caudal regions contained labeled cells in both the medial and lateral poles.     

Viscertopic localization of preganglionic parasympathetic cell bodies of origin of the anterior and posterior subdiaphragmatic vagus nerves

Utilizing the retrograde HRP transport method, fibers from anterior and posterior subdiaphragmatic branches of the vagus nerve in the rat were traced to their cells of origin in the brainstem. Efferents to the gut supplied by the subdiaphragmatic vagus nerves derive from cell bodies organized in a viscerotopic, spindle-shaped longitudinal cell column throughout the longitudinal extent of the classically described dorsal nucleus of the vagus (DNV) and in regions of nucleus commissuralis (NC), caudal to the DNV. This entire longitudinal group of cells is called the DNV cell column. In the caudal one third of the DNV cell column, the cell bodies were found in the midline and paramedian posterior portion of the NC, and in the anterior portion of the caudal DNV, in a horizontally oriented cluster of cells when viewed in cross section. In the middle one third of the DNV cell column, the cell bodies moved laterally, but still maintained their anterior position in the nucleus. In the rostral one third of the cell column, the cell bodies were located at the lateral margin of the DNV. A few scattered cell bodies extended caudally from the DNV cell column into the dorsal region of lamina X of spinal cord, and reached as far caudal as the C₅-C₆ segments. The anterior subdiaphragmatic branch of the vagus contained axons whose cell bodies were mainly but not exclusively located in the ipsilateral (left) side of the medulla, while the posterior subdiaphragmatic branch of the vagus contained axons whose cell bodies were found bilaterally in the medulla, with a majority (approx. 60%) located on the ipsilateral (right) side, and approximately 40% located on the contralateral (left) side.     

Vagus nerve afferent and efferent innervation of the rat uterus: An electrophysiological and HRP study

To determine a possible brainstem connection with the uterus, a study with electrophysiological techniques and horseradish peroxidase (HRP) tracing was performed in the rat. Neurons of the nucleus of the tractus solitarius decreased in discharge frequency during cervicovaginal distension. HRP injections into the uterine walls resulted in the appearance of labelled cells in the nodose ganglion and in the dorsal motor nucleus of the vagus nerve. The results demonstrate a direct bidirectional vagal complex-uterus connection via the vagus nerve. Results are discussed in terms of a complex uterus control system in which the paraventricular nucleus might play an integrative role.     

Pituitary adenylate cyclase activating polypeptide-immunoreactive sensory neurons innervate rat adrenal medulla

Rat adrenal chromaffin cells were invested by a dense network of nerve fibers immunoreactive to pituitary adenylate cyclase activating polypeptide-38 (PACAP-IR). Immunohistochemical studies demonstrated the presence of PACAP-IR in nodose and dorsal root ganglion cells, but not in neurons of the intermediolateral cell column and other autonomic nuclei of the thoracic and upper lumbar spinal cord. Somata of the T7 to T12 paravertebral ganglia were PACAP-negative. A few lightly labeled neurons were occasionally noted in the dorsal motor nucleus of the vagus. Injection of the retrograde tracer Fluorogold into the left adrenal medulla 3 days prior to sacrifice resulted in the labeling of a population of neurons in the ipsilateral spinal cord intermediolateral cell column (T1 to L1), ipsilateral and contralateral nodose ganglia and ipsilateral dorsal root ganglia from T7 to T10 inclusive. A small number of lightly labeled somata was occasionally noted in the dorsal motor nucleus of the vagus. Combined retrograde tracing and PACAP immunohistochemistry showed that a population of Fluorogold-containing nodose and dorsal root ganglion cells were also PACAP-positive. Pre-treatment of the rats with capsaicin caused a marked reduction of the PACAP-IR in the adrenal gland as well as in the superficial layers of the dorsal horn and caudal spinal trigeminal nucleus. These findings, in conjunction with the apparent absence of PACAP-IR in spinal sympathetic preganglionic neurons, sympathetic postganglionic neurons, and dorsal motor nucleus of the vagus, raise the possibility that PACAP-IR fibers observed in the adrenal medulla are primarily sensory in origin. As a corollary, catecholamine secretion from chromaffin cells may be modulated by the peptidergic sensory afferents in addition to the cholinergic sympathetic preganglionic nerve fibers.     

Ultrastructural identification of labeled neurons in the dorsal motor nucleus of the vagus nerve following injections of horseradish peroxidase into the vagus nerve and brainstem

The efferent connections of two types of neurons in the dorsal motor nucleus of the vagus nerve (DMV) were studied in the cat by light and electron microscopy following horseradish peroxidase (HRP) injections into the cervical vagus nerve or brainstem. After injections of HRP into the vagus nerve, up to 80% of medium-sized neruons averaging 26 × 20 μm in 1-μm-thick sections were retrogradely labeled while no small neurons were labeled in the DMV. Incubation with either diaminobenzidene (DAB) or p-phenylenediamine-pyrocatechol (PPD-PC) chromogens yielded electron-dense reaction products localized mainly in lysosomes. Identification of label at the ultrastructural level was facilitated by omitting lead citrate staining and by counting numbers of lysosomes, which were higher in labeled neurons. Quantitative comparisons of the dimensions of labeled and unlabeled somata demonstrated that retrograde transport and incorporation of HRP had no effect on cell size within the 2–3-day survival times used in this study. In order to determine whether neurons in the DMV project to higher levels of the brain stem, large injections of HRP (1–3 μl) were made into the pons, mesencephalon, hypothalamus, and amygdala. After injections of HRP into the brainstem, only small neurons, measuring 17 × 10 μm, were retrogradely labeled. Approximately 90% of the small neurons remained unlabeled following the HRP injections. The ultrastructrual features of the labeled small neurons included an invaginated nucleus, low cytoplasmic/nuclear ratio, and relatively fewer organelles than the medium-sized neurons. A quantitative analysis of labeled and unlabeled small neurons demonstrated that the labeled neurons were significantly larger than the unlabeled small neurons. Thus, two populations of small neurons may exist in the DMV. One population appears to have ascending projections to higher levels of the brainstem while the other more numerous population may be interneurons or project for only short distances.