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.     

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.     

Medullary origin of vagal preganglionic axons to the heart of the cat

It is apparent from the literature that a controversy exists concerning the site of origin of cardiac vagal preganglionic axons. Physiological studies have suggested that the location of these neurons may be different in different species and there has been disagreement between physiological and anatomical findings in the same species. We now present anatomical and neurophysiological studies suggesting that in the cat cardiac vagal preganglionic neurons are located in two medullary regions: the areas of the dorsal motor nucleus of the vagus (DMV) and of the nucleus ambiguus (AMB). This suggestion is based on the following observations. Firstly, after application of horseradish peroxidase to the right cardiac branches of the vagus nerve, labeled neurons were found primarily in the regions of te DMV and AMB. Additional scattered neurons were found in the reticular formation between these two nuclei. Secondly, following injections of tritiated amino acids into either the DMV or AMB, labeled vagal fibers were found in the atrial myocardium. Finally, electrical stimulation of the right cardiac branches of the vagus nerve antidromically activated DMV or AMB, labeled vagal fibers were found in the atrial myocardium. Finally, neurons in the DMV and AMB regions with latencies corresponding to conduction velocities of B-fibers. In addition, these neurons were orthodromically excited by electrical stimulation of the carotid sinus and aortic depressor nerves.     

Brainstem origin of preganglionic cardiac motoneurons in the muskrat

The muskrat, an aquatic rodent with a brisk and reliable diving response, shows a remarkable bradycardia after nasal stimulation. However, the medullary origin of cardiac preganglionic motoneurons is unknown in this species. We injected fat pads near the base of the heart of muskrats with a WGA-HRP solution to label retrogradely preganglionic parasympathetic neurons that project to the cardiac plexi. Results showed that the preponderance of labeled neurons was in ventrolateral parts of the medulla from 1.5 mm caudal to the obex to 2.0 mm rostral. Eighty-nine percent of the labeled neurons were located bilaterally in the external formation of the nucleus ambiguus, 5.6% were in the lateral extreme of the dorsal motor nucleus of the vagus nerve and 5.3% were found in the intermediate area in between these two nuclei. Although controversy still exists concerning the medullary origin of preganglionic cardiac motoneurons, our results from muskrats agree with those from most other species where preganglionic cardiac motoneurons were located just ventral to the nucleus ambiguus.     

Different astroglial reaction between the vagal dorsal motor nucleus and nucleus ambiguus following vagal-hypoglossal nerve anastomosis in cats

The dorsal motor nucleus of the vagus (DMV) and nucleus ambiguus (NA) were both traced with horseradish peroxidase (HRP) retrograde labelling technique after vagal-hypoglossal nerve anastomosis (VHA). By light microscopy, reinnervation of the new target, viz. tongue skeletal musculature, by DMV and NA was established at 22 days postoperation (dpo) as shown by the neuronal labelling with HRP. Ultrastructurally, signs of retrograde degeneration occurred in some DMV and NA neurons between 3 and 25 days after VHA. The incidence of darkened dendrites, an early sign of dendritic loss, was more common in the DMV compared to the NA. Accompanying the neuronal alteration were drastic astrocytic reactions in the DMV, but not in the NA. Between 3 and 7 dpo, the astrocytes in the DMV showed extensively hypertrophied processes and by 22 dpo, the somata and dendrites of HRP-labelled DMV neurons, but not NA's, appeared to be delineated by the increased lamellar astrocytic processes. Such a feature was sustained throughout the remaining postoperative intervals up to 500 dpo. It is concluded that the DMV motoneurons being autonomic in nature are probably not conducive to the newly acquired target organ. Hence, the insulation of the regenerating DMV motoneurons by the astroglial ensheathment would be vital in the neuronal remodelling and reconstruction of the vagal-hypoglossal pathway.     

Cardiotopic organization of the nucleus ambiguus? An anatomical and physiological analysis of neurons regulating atrioventricular conduction

Previous data indicate that there are anatomically segregated and physiologically independent parasympathetic postganglionic vagal motoneurons on the surface of the heart which are capable of selective control of sinoatrial rate, atrioventricular conduction and atrial contractility. We have injected a retrograde tracer into the cardiac ganglion which selectively regulates atrioventricular conduction (the AV ganglion). Medullary tissues were processed for the histochemical detection of retrogradely labeled neurons by light and electron microscopic methods. Negative dromotropic retrogradely labeled cells were found in a long column in the ventrolateral nucleus ambiguus (NA-VL), which enlarged somewhat at the level of the area postrema, but reached its largest size rostral to the area postrema in an area termed the rostral ventrolateral nucleus ambiguus (rNA-VL). Three times as many cells were observed in the left rNA-VL as compared to the right (P < 0.025). Retrogradely labeled cells were also consistantly observed in the dorsal motor nucleus of the vagus (DMV). The DMV contained one third as many cells as the NA-VL. The right DMV contained twice as many cells as the left (P < 0.05). These data are consistent with physiological evidence that suggests that the left vagus nerve is dominant in the regulation of AV conduction, but that the right vagus nerve is also influential. While recording the electrocardiogram in paced and non-paced hearts,l-glutamate (GLU) was microinjected into the rNA-VL. Microinjections of GLU caused a 76% decrease in the rate of atrioventricular (AV) conduction (P < 0.05) and occasional second degree heart block, without changing heart rate. The effects of GLU were abolished by ipsilateral cervical vagotomy. These physiological data therefore support the anatomical inference that CNS neurons that are retrogradely labeled from the AV ganglion selectively exhibit negative dromotropic properties. Retrogradely labeled negative dromotropic neurons displayed a round nucleus with ample cytoplasm, abundant rough endoplasmic reticulum and the presence of distinctive somatic and dendritic spines. These neurons received synapses from afferent terminals containing small pleomorphic vesicles and large dense core vesicles. These terminals made both asymmetric and symmetric contacts with negative dromotropic dendrites and perikarya, respectively. In conclusion, the data presented indicate that there is a cardiotopic organization of ultrastructurally distinctive negative dromotropic neurons in the NA-VL. This central organization of parasympathetic preganglionic vagal motoneurons mirrors the functional organization of cardioinhibitory postganglionic neurons of the peripheral vagus nerve. These data are further discussed in comparison to a recent report on the light microscopic distribution and ultrastructural characteristics of negative chronotropic neurons in the NA-VL⁴². The data support the hypothesis that anatomically separated and functionally selective parasympathetic preganglionic vagal motoneurons in the NA may independently control AV conduction and cardiac rate.     

Different astroglial reaction between the vagal dorsal motor nucleus and nucleus ambiguus following vagal–hypoglossal nerve anastomosis in cats

The dorsal motor nucleus of the vagus (DMV) and nucleus ambiguus (NA) were both traced with horseradish peroxidase (HRP) retrograde labelling technique after vagal–hypoglossal nerve anastomosis (VHA). By light microscopy, reinnervation of the new target, viz. tongue skeletal musculature, by DMV and NA was established at 22 days postoperation (dpo) as shown by the neuronal labelling with HRP. Ultrastructurally, signs of retrograde degeneration occurred in some DMV and NA neurons between 3 and 25 days after VHA. The incidence of darkened dendrites, an early sign of dendritic loss, was more common in the DMV compared to the NA. Accompanying the neuronal alteration were drastic astrocytic reactions in the DMV, but not in the NA. Between 3 and 7 dpo, the astrocytes in the DMV showed extensively hypertrophied processes and by 22 dpo, the somata and dendrites of HRP-labelled DMV neurons, but not NA’s, appeared to be delineated by the increased lamellar astrocytic processes. Such a feature was sustained throughout the remaining postoperative intervals up to 500 dpo. It is concluded that the DMV motoneurons being autonomic in nature are probably not conducive to the newly acquired target organ. Hence, the insulation of the regenerating DMV motoneurons by the astroglial ensheathment would be vital in the neuronal remodelling and reconstruction of the vagal–hypoglossal pathway.     

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.     

Localization of cardiac parasympathetic preganglionic neurons in the medulla oblongata of pigeon, Columba livia: a study using fragment C of tetanus toxin.

The binding fragment of tetanus toxin, fragment C, was injected into several different regions of the pigeon heart. Retrogradely and/or transneuronally labeled cardiomotor parasympathetic preganglionic neurons were found in two separate nuclei within the medulla oblongata. The majority of fragment C-immunolabeled cells was confined to the caudal division of the nucleus ambiguus. This nuclear region is likely to be homologous to the ventrolateral nucleus of the external formation of the nucleus ambiguus in mammals. A smaller fraction (10-30%) of fragment C-positive cardiomotor preganglionic neurons were localized within a restricted portion of the ventrolateral subnucleus of the dorsal motor nucleus of the vagus nerve. This dual cardiac representation in an avian is very similar to the organization established in several mammalian species, and suggests that the brainstem organisation of cardiac parasympathetic efferents is evolutionarily stable across avians and mammals.     

Innervation of the rat trachea by bilateral cholinergic projections from the nucleus ambiguus and direct motor fibers from the cervical spinal cord: a retrograde and anterograde tracer study

A tract-tracer method was employed to examine the innervation of the rat trachea. Cholera toxin beta subunit (CTB) was injected into the following locations in separate groups of rats: (1) ventral trachea, (2) lateral trachea, (3) ventral trachea after the excision of the nodose ganglion, and (4) ventral trachea after the transection of C1-C2 spinal nerves. CTB injection in the ventral trachea showed bilateral labeling of neurons in the nucleus ambiguus (NA), medial subnucleus of the nucleus of the solitary nucleus, dorsal motor nucleus of the vagus (DMV), and lamina IX of C1-C6. CTB injection in the lateral trachea showed significant ipsilateral predominance of neuronal labeling in the NA and lamina IX of C1-C2 segments. CTB injection in rats after the excision of the nodose ganglion revealed no labeling in the ipsilateral DMV and NA and a significant reduction of neuronal labeling in C1. CTB injection in rats after the transection of C1-C2 spinal nerves showed a significant decrease in the number of labeled neurons in ipsilateral NA, C1, and C2 and no labeling of fibers in C1-C2. The combination of retrograde fluorogold labeling and choline acetyltransferase (ChAT) immunostaining revealed that all fluorogold-labeled neurons in the NA and lamina IX of C1-C2 colocalized with ChAT. The injection of biotinylated dextran amine in NA produced labeling in axonal terminals on postganglionic neurons, but not in other regions of the trachea. Our findings indicate that the rat trachea is innervated bilaterally by cholinergic motor neurons in NA and C1-C2, while those traveling through the spinal nerves project directly to the trachea.     

Substance P nerve terminals synapse upon negative chronotropic vagal motoneurons

Previous data indicate that there are anatomically segregated and physiologically independent parasympathetic ganglia on the surface of the heart which are capable of selective control of sino-atrial rate, atrio-ventricular conduction, and atrial contractility. We have injected a retrograde tracer into the cardiac ganglion which selectively regulates heart rate (the SA ganglion). Medullary tissues were processed for the histochemical visualization of retrogradely labeled neurons and for the immunohistochemical detection of the neurotransmitter substance P (SP) by dual labeling light and electron microscopic methods. Negative chronotropic retrogradely labeled cells were found in a long slender column in the ventrolateral nucleus ambiguus (NA-VL) which enlarged somewhat at the level of the area postrema. These cells were found bilaterally, but they were asymmetrically distributed. Half the animals showed a pronounced right side predominance in retrograde labeling, while the other half of the animals showed a lesser left side predominance. These observations may help to explain some of the controversy in the literature concerning the relative influence of the right and left vagus nerves on sinus rate. Ultrastructural examination demonstrated axo-somatic and axo-dendritic contacts between SP nerve terminals and retrogradely labeled negative chronotropic NA-VL neurons. SP immunoreactivity was often associated with large dense-core vesicles in terminals forming either symmetric or asymmetric synapses. These observations provide a potential anatomical substrate for the centrally mediated bradycardia elicited by microinjections of SP into the NA. SP immunoreactive terminals were also observed to make axo-somatic, axo-dendritic, and axo-axonic synapses with unlabeled neurons in NA-VL. These data suggest that SP may also modulate the activity of other vagal preganglionic neurons.     

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.     

Projections from the nucleus tractus solitarii to the rostral ventrolateral medulla

Projections from the nucleus tractus solitarii (NTS) to autonomic control regions of the ventrolateral medulla, particularly the nucleus reticularis rostroventrolateralis (RVL), which serves as a tonic vasomotor center, were analyzed in rat by anterograde, retrograde, and combined axonal transport techniques. Autonomic portions of the NTS, including its commissural, dorsal, intermediate, interstitial, ventral, and ventrolateral subnuclei directly project to RVL as well as to other regions of the ventrolateral medulla. The projections are organized topographically. Rostrally, a small cluster of neurons in the intermediate third of NTS, the subnucleus centralis, and neurons in proximity to the solitary tract selectively innervate neurons in the retrofacial nucleus and nucleus ambiguus. Neurons generally located in more caudal and lateral sites in the NTS innervate the caudal ventrolateral medulla (CVL). The RVL, CVL, and nucleus retroambiguus are interconnected. A combined retrograde and anterograde transport technique was developed so as to prove that projections from the NTS to the ventrolateral medulla specifically innervate the region of RVL containing neurons projecting to the thoracic spinal cord or the region of the nucleus containing vagal preganglionic neurons. When the retrograde tracer, fast blue, was injected into the thoracic spinal cord, and wheat germ agglutinin-conjugate horseradish peroxidase (HRP) was injected into the NTS, anterogradely labeled terminals from the NTS surrounded the retrogradely labeled neurons in the RVL and in the nucleus retroambiguus in the caudal medulla. Among the bulbospinal neurons in the RVL innervated by the NTS were adrenaline-synthesizing neurons of the C1 group. When fast blue was applied to the cervical vagus, and HRP was injected into the NTS, anterogradely labeled terminals from the NTS surrounded retrogradely labeled neurons in the rostral dorsal motor nucleus of the vagus, the region of the nucleus ambiguus, the retrofacial nucleus, and the dorsal portion of the RVL, a region previously shown to contain cardiac vagal preganglionic neurons. This combined anterograde and retrograde transport technique provides a useful method for tracing disynaptic connections in the brain. These data suggest that the RVL is part of a complex of visceral output regions in the ventrolateral medulla, all of which receive afferent projections from autonomic portions of the NTS. Bulbospinal neurons in the RVL, in particular the C1 adrenaline neurons, may provide a portion of the anatomic substrate of the baroreceptor and other visceral reflexes.     

Collaterals of recurrent laryngeal nerve fibres innervate the thymus: a fluorescent tracer and HRP investigation of efferent vagal neurons in the rat brainstem

The origin and course of efferent vagal fibers, which innervate the rat thymus, were investigated by a fluorescent retrograde double labeling method, using Fast blue (FB) and Diamidino yellow dihydrochloride (DY) as tracers. In the same animal, one tracer was injected into the cranial portion of the right lobe of the thymus and the other dye was deposited around the cut end of the right recurrent laryngeal nerve. The neuronal population giving origin to the recurrent nerve was mapped by using retrograde labeling with HRP applied to the central stump of the nerve. The HRP retrograde axonal transport showed that most efferent vagal fibers of the recurrent nerve have their perikarya in the nucleus retroambigualis (NRA), nucleus ambiguus (NA), and to a lesser extent in the nucleus retrofacialis (NRF). In fluorescent retrograde double labeling of thymus and recurrent laryngeal nerve both single and double labeled cells were found. The cells labeled by the injections into the thymus were colocalized with the neurons labeled by the tracer deposited in the recurrent laryngeal nerve to the NRA, NA, and NRF. Moreover along the rostrocaudal extent of the NRF and NA double labeled cells were present, showing that some of the thymic efferents are collaterals of the recurrent nerve fibers. Our experiments shown that some thymic vagal fibres originate from neurons of nucleus dorsalis nervi vagi (NDV) as demonstrated both by HRP and FB injected thymuses. The possible role of these efferents in thymic function is briefly discussed.     

The nucleus of the solitary tract in the monkey: projections to the thalamus and brain stem nuclei

The projections of the nucleus of the solitary tract (NST) were studied by autoradiographic anterograde fiber-tracing and horseradish peroxidase (HRP) retrograde cell-labeling. Tritiated proline and leucine were deposited in electrophysiologically identified regions of NST. Injections of NST at levels caudal to where the vagus enters the nucleus, from which responses were evoked by stimulation of cranial nerves IX and X, revealed topographically organized bilateral projections to, most prominently, the ventrolateral medullary reticular formation which contains neurons of the ambiguus complex, and to the lateral and medial parabrachial nuclei, including a small portion of the medially adjacent central gray substance. Labeled fibers in the ventrolateral reticular formation were present from the nucleus retroambigualis rostralward to the retrofacial nucleus, with the densest concentration located over the nucleus ambiguus proper. The parabrachial projection was confirmed using HRP and shown to originate from cells in the medial subdivision of NST. Due to the problem of fibers en passant, it was not possible to interpret conclusively the cell-labeling seen around the solitary tract after HRP injections made in the region of the nucleus ambiguus. Labeled fibers were also traced from caudal NST to the dorsal motor nucleus of the vagus, but their origin could not be determined with certainty. Other labeled axons, traced to circumscribed parts of the inferior olivary complex and via the contralateral medial lemniscus to VPL of the thalamus, were shown in HRP experiments to originate from the dorsal column nuclei rather than NST. No labeled fibers were traced into the spinal cord, nor were any cells labeled in NST after large HRP deposits in upper cervical segments. Isotope deposits at levels of NST rostral to the entrance of the vagus, from which responses were evoked by rapid stimulation of the tongue, revealed an ipsilateral projection which ascends as a component of the central tegmental tract to the parvicellular part of the ventral posteromedial thalamic nucleus (VPMpc). After small HRP deposits in VPMpc, labeled cells in NST were restricted to the rostral part of the lateral subdivision. No labeled axons were traced from rostral NST to the ambiguus complex or parabrachial area. Injections of 3H-amino acids at intermediate levels of NST resulted in fiber-labeling in VPMpc, the parabrachial area, and the ambiguus complex.     

The location of extrinsic efferent and afferent nerve cell bodies of the normal canine stomach

The location of the extrinsic efferent and afferent nerve cell bodies to the mucosa, submucosa, and tunica muscularis of the cardiac, gastric, and pyloric gland regions of the ventral stomach and to the mucosa-submucosa alone of these 3 glandular gastric regions was determined using the horseradish peroxidase technique. All animals of the study demonstrated labeling bilaterally in the rostrocaudal extent of the dorsal motor nucleus of the vagus nerve (DMV) although mucosa-submucosa injections resulted in fewer labeled cells in the DMV. There was no evidence of viscerotopic organization within the DMV for the different gastric regions. However, the left nucleus generally contained a greater number of labeled cells than the right nucleus. Injection of the mucosa, submucosa, and tunica muscularis of the cardiac gland region also resulted in labeling in the nucleus ambiguus in 4 of 5 animals. The vast majority of labeled postganglionic sympathetic neurons were found in the celiacomesenteric ganglion. Labeled cells were also located variously in the stellate ganglion, middle cervical ganglion, and sympathetic trunk ganglia for the different groups. There was no discernible pattern of localization of labeled cells within a sympathetic ganglion. For the stomach, afferent labeled cells were located in the range of the first thoracic to fourth lumbar spinal ganglia and the nodose ganglia, bilaterally. As with sympathetic neurons, there was no discernible pattern of localization of labeled cells within a sensory ganglion.     

Localization of vagal cardioinhibitory preganglionic neurons with rat brain stem.

In chloralose-urethane anesthetized spinal rats, electrical stimulation of systematically chosen points over the entire caudal brain stem area was carried out to explore the site(s) responsible for vagally mediated bradycardia. A dorsomedial locus including the nucleus dorsalis and the adjacent structures, the nucleus tractus solitarius, the nucleus commissuralis and the area postrema, and a ventrolateral locus around the nucleus ambiguus were found to elicit bradycardia with low threshold and high responsiveness. In another series of experiments, horseradish peroxidase (HRP) was iontophoretically administered through a glass capillary microelectrode into the identified cardiac branch of the vagus nerve of rats in order to localize more precisely the cells of origin of vagal cardioinhibitory fibers within the brain stem. Distribution of the HRP-labeled cells was not confined to one area, but these cells were found within the nucleus dorsalis, the reticular formation surrounding the nucleus ambiguus and an intermediary zone between the two nuclei. Such a pattern of distribution of vagal cardioinhibitory preganglionic cells is discussed in relation to phylogenetic and ontogenetic development of the vagal motor nuclei.     

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.     

A cardioinhibitory area in the midbrain central tegmental field of cats

A cardioinhibitory area in the central tegmental field of the midbrain (CIM) was studied in cats under chloralose-urethane anesthesia. Electrical and chemical (glutamate and DL-homocysteic acid) stimulations in this area produced marked bradycardia accompanied by mostly hypotension or minimal change in arterial pressure and by occasional hypertension particularly in the dorsal portion. CIM excitation potentiated the reflex bradycardia induced by IV phenylephrine, while bilateral electrolytic lesion of CIM neither changed the resting cardiovascular parameters nor the reflex bradycardia. The CIM bradycardia was not affected by supracollicular decerebration, but substantially reduced by unilateral vagotomy and completely eliminated by bilateral vagotomy. Destruction of the ambiguus nucleus (NA) and solitary and dorsal motor nuclei (NTS/DMV) abolished the bradycardia. Midline bisection at the midbrain-pontine level only slightly reduced the bradycardia while at the medullary level it was moderately attenuated. Electrolytic lesion of the cardioinhibitory area in gigantocellular reticular nucleus (GRN) abolished the bradycardia. These findings suggest that CIM is an independent mechanism which may send axons to GRN from which the axons may in turn synapse with the NTS/DMV complex and NA. Its final output may utilize both vagus nerves to modulate baroreceptor reflex in promoting bradycardia.     

Origins of cardiac vagal preganglionic fibers: A retrograde transport study

The origin of cardiac preganglionic neurons in the rat was investigated using the retrograde transport of horseradish peroxidase (HRP). A single injection of HRP was made into the right myocardium in either a sinoatrial or mid-ventricular location. Labeled cells were found in the mid- and lower medulla primarily in and around the nucleus ambiguus (NA) 600–1800 μm above the obex. The dorsal motor nucleus of X (DMN) was sparsely labeled and a few cells were found in an intermediate zone near the level of the obex. Labeling was bilateral with slightly heavier labeling found ipsilateral to the injection site than contralateral to it. Following a unilateral vagotomy, labeled cells were only found ipsilateral to the intact vagus. Atrial and midventricular injections yielded similar results. Occasionally only 1– cells in the NA were labeled per section. Inspection of serial sections revealed that in these sparsely labeled rats, the HRP was often in the same location within the NA forming a column of cells within the nucleus. The columns sometimes extended at least 240 μm in the rostral-caudal direction. The columnar organization was most apparent in rats with few labeled cells presumably because it was obscured in nuclei that were heavily labeled.In a second group of rats, the right vagus was cut at the cervical level and dipped in HRP to determine the extent of the NA and DMN in rats. In these animals, heavier labeling was found in the DMN than in the NA. Cells in the DMN were filled from the upper spinal cord to its most rostral extent 1200 μm above the obex. Thus, although the DMN and NA send projections in the vagus nerve, those axons terminating in the myocardium primarily originate in the NA.