Axonal branching of canine sympathetic postganglionic cardiopulmonary neurons. A retrograde fluorescent labeling study

In 11 dogs fluorescent retrograde tracers were injected into physiologically identified left-sided sympathetic cardiopulmonary nerves. When two different ipsilateral cardiopulmonary nerves were injected, labeled cells from each injected nerve had overlapping distributions in the middle cervical and stellate ganglia. Most retrogradely labeled neurons were located in the middle cervical ganglion and cranial pole of the stellate ganglion. Following the injection of two different tracers into two different nerves, some neurons in the middle cervical ganglion were retrogradely labeled with two tracers. Double-labeled neurons were rarely found in the stellate ganglion. There were areas within the ganglia in which labeled neurons projected predominantly to one cardiopulmonary nerve. In the thoracic autonomic nervous system Fast Blue was transported most effectively. Bisbenzimide was not transported as well as Fast Blue and Nuclear Yellow was very poorly transported in cardiopulmonary nerves. The results demonstrate that some efferent postganglionic sympathetic neurons project axons into at least two different cardiopulmonary nerves and that an anatomical substrate for axo-axonal reflexes exists in the thoracic sympathetic nervous system.     

Localization of efferent sympathetic neurons and afferent neurons in dorsal root ganglia innervating the heart

The retrograde transport of horseradish peroxidase (HRP) has been used to study the localization and the number of neurons innervating the heart in the right stellate ganglion and accessory cervical ganglion, spinal cord and dorsal root ganglia of the cat. HRP was applied to the central cuts of anastomose of the stellate ganglion with the vagal nerve, of the vagosympathetic trunk caudal to anastomose and of the inferior cardiac nerve. HRP-labelled neurons were detected in the stellate ganglion in the regions which give off nerves, whereas in the accessory cervical ganglion labelled neurons were distributed throughout the whole ganglion. HRP-stained cells were found in the anastomose. In the spinal cord labelled neurons were detected in the lateral horn of T1-T5 segments. In the dorsal root ganglion the greatest number of neurons was observed in T2-T4 segments.     

Evidence for a sympathetic component in motor branches of the facial nerve: a horseradish peroxidase study in the cat

The retrograde transport of horseradish peroxidase (HRP) was used to examine the location of sympathetic ganglion cells with axons in facial motor branches of the cat. Large numbers of HRP-labeled neurons were observed in the rostro-anterior part of the superior cervical ganglion. In addition, some labeled neurons were found in the cervical sympathetic trunk, the accessory cervical, middle cervical and stellate ganglia.     

Horseradish peroxidase localization of the sympathetic postganglionic neurons innervating the cat heart

The localization of the sympathetic postganglionic neurons innervating the cat heart has been investigated by using retrograde axonal transport of horseradish peroxidase (HRP). HRP was injected into the subepicardial layers of 4 different cardiac regions. The animals were sacrificed 72-96 h later and fixed by perfusion via the left ventricle. The paravertebral sympathetic ganglia from the superior cervical, middle cervical and stellate ganglia to T10 ganglia were removed and processed for HRP identification. Following injections of HRP into the apex of the heart, the sinoatrial (SA) nodal region and the ventral wall of the right ventricle, we observed that HRP-labeled sympathetic neurons were localized predominantly in the right stellate ganglia, and to a lesser extent, in the right superior and middle cervical ganglia, and left stellate ganglia. Fewer labeled cells were found in the right T4-T6. T8 and T9. After HRP injection into the dorsal wall of the left ventricle, HRP-labeled cells were present mainly in the left stellate ganglia.     

Ganglionic distribution of afferent neurons innervating the canine heart and cardiopulmonary nerves

The ganglionic distribution of the perikarya of afferent axons in cardiopulmonary nerves or the heart was studied in 64 dogs by injecting horseradish peroxidase into physiologically identified cardiopulmonary nerves or different regions of the heart. In 6 additional dogs, horseradish peroxidase was injected into the aortic arch, pericardial sac, left ventricular cavity or the skin. After injections into cardiopulmonary nerves, retrogradely labeled perikarya were found in the ipsilateral nodose ganglion and the ipsilateral C7-T7 dorsal root ganglia. After injections into different regions of the heart, retrogradely labeled neurons were found in the nodose ganglia bilaterally and in the C6-T6 dorsal root ganglia bilaterally. Many more retrogradely labeled neurons were found in the nodose ganglia in comparison to the dorsal root ganglia. The largest numbers of retrogradely labeled perikarya in the dorsal root ganglia occurred in the T 2-4 ganglia following nerve or heart injections. Following injections into specific regions of the heart or individual physiologically identified cardiopulmonary nerves, regional distributions of labeled neurons could not be identified within or among ganglia with respect to the structures injected. Perikarya in dorsal root ganglia which were labeled after heart injections ranged in area from 436-3280 microns 2 (X = 1279 +/- 51 S.E.M.) while after skin injections labeled perikarya ranged in area from 224-5701 microns 2 (X = 1631 +/- 104 S.E.M.). The results show that the afferent innervation of the canine heart is provided by neurons located throughout the nodose ganglia and to a lesser degree in the C6-T6 dorsal root ganglia bilaterally. The bilateral distribution of cardiac afferent neurons raises questions regarding mechanisms underlying unilateral symptoms frequently associated with heart disease.     

Sympathetic postganglionic innervation of the cardiac coronary artery in cats

The localization of the sympathetic postganglionic neurons innervating the cardiac coronary arteries of the cat was investigated using retrograde axonal transport with horseradish peroxidase. We found after the enzyme was applied to the main trunk of the right coronary artery, and to the main trunk and the terminal branch of the ventral descending vessels of the left coronary artery, the peroxidase-labeled sympathetic neurons were localized predominantly in the right stellate ganglia, with a few cells in the left stellate ganglia. There were very few labeled cells in the middle cervical, superior cervical, and T4-7 ganglia on both sides. After peroxidase application to the terminal branch of the dorsal descending vessels of the right coronary artery, labeled cells were mainly in the left stellate ganglia, with only a few cells in the right stellate ganglia.     

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.     

Distribution of neuropeptide-like immunoreactivity in intact and chronically decentralized middle cervical and stellate ganglia of dogs

Neuropeptide-like immunoreactivity to antisera raised against Leu- and Met-enkephalin, vasoactive intestinal peptide (VIP), neuropeptide Y (NPY) and substance P (SP) have been studied immunohistochemically in middle cervical and stellate ganglia of dogs. To investigate the relationship of the peptides to one another as well as to preganglionic and postganglionic neurons, intact and chronically decentralized middle cervical and stellate ganglia were studied. Ganglia were processed for immunohistochemistry in unoperated dogs and in dogs two weeks after unilateral ganglionic decentralization. The immunoreactivity for each peptide had a characteristic distribution in the ganglia. These distributions differed from one another and from the distribution of cardiac postganglionic sympathetic neurons. Camera lucida drawings of peptide distributions were made to compare different peptides and counts were made to determine the percentages of cells immunoreactive for a given peptide. The results demonstrated that enkephalin-like immunoreactivity in axons was present in both the stellate and middle cervical ganglia, but was heaviest in the caudal 2/3 of the stellate ganglia. Enkephalin-like immunoreactive fibers formed pericellular baskets around stellate ganglion neurons. VIP-like immunoreactive cell bodies and processes were distributed sparsely, but widely, in the stellate ganglia and to a lesser extent in the middle cervical ganglia. One of two commercial antisera to SP resulted in immunoreactive staining of cell bodies and processes in the stellate ganglia. SP-like immunoreactivity in neurons represented about 10% or less of the cells in the stellate ganglia. At least 80-85% of the neurons in the stellate and middle cervical ganglia were immunoreactive for NPY antisera. Decentralization eliminated enkephalin-like immunoreactive staining in the middle cervical and stellate ganglia, but not the VIP-, NPY- and SP-like immunoreactive staining of neurons in these ganglia. In summary, the enkephalin-like immunoreactive axons in the thoracic autonomic ganglia appear to be derived from extrinsic neurons, most likely from preganglionic spinal neurons. VIP-, SP- and NPY-like immunoreactivity were not significantly affected by decentralization. The results provide anatomical evidence for substrates related to neuropeptidergic synaptic mechanisms in thoracic autonomic ganglia.     

Negative inotropic vagal preganglionic neurons in the nucleus ambiguus of the cat: neuroanatomical comparison with negative chronotropic neurons utilizing dual retrograde tracers

The vagal postganglionic controls of cardiac rate and left ventricular contractility are mediated by separate intracardiac ganglia, the sino-atrial (SA) and cranio-ventricular (CV) ganglia, respectively. We injected a different retrograde tracer into each of these ganglia (in the same animal) and subsequently examined the brain for the presence of single labeled or double labeled vagal preganglionic neurons. Retrogradely labeled cells from either ganglion were found exclusively in the ventrolateral nucleus ambiguus (NA-VL). There was considerable overlap in the distribution of labeled cells from either ganglion, however fewer than 3% of labeled neurons were double labeled. The data are consistent with the hypothesis that the preganglionic controls of cardiac rate and left ventricular contractility are mediated by largely separate but overlapping groups of cardioinhibitory neurons originating from the NA-VL. These neurons have parallel but morphologically independent pathways projecting to the SA and CV ganglia. Physiological experiments are needed to support this hypothesis.     

Origin of sympathetic and sensory innervation of the elbow joint in the rat: A retrograde axonal tracing study with wheat germ agglutinin conjugated horseradish peroxidase

After injection of wheat germ agglutinin conjugated horseradish peroxidase (WGA-HRP) into the elbow joint of adult rats, labeled neurons were found in the stellate and the T2-T4 ganglia of the ipsilateral sympathetic trunk, and also in dorsal root ganglia at the C4–T4 levels. Most labeled sympathetic cells, 90% or more, were located in the stellate ganglion. The sensory innervation to the joint originated mainly from the dorsal root ganglia at the levels of C7–T1.     

Afferent innervation of the lower oesophageal sphincter of the cat. An HRP study

Labeling of afferent neurons by the retrograde axonal transport of horseradish peroxidase (HRP) was performed on anaesthetized cats in order to examine the afferent innervation of the lower oesophageal sphincter (LOS), involving both the vagal and the sympathetic nerves. The labeled cells, whose fibres follow the sympathetic pathways were found in dorsal root ganglia from T1 to L2. Nerve section experiments indicated that the main pathways involved were the splanchnic nerves, as expected from classical data. Additional pathways passing through the sympathetic cardiac branch emerging from the stellate ganglion and the thoracic sympathetic branches were also evidenced. This work corroborated the electrophysiological data showing the richness of the LOS sensory vagal innervation. Nevertheless, in this case the difficulties related to the HRP technique are particularly enhanced since the abdominal sensory vagal fibres can be affected by HRP injections.     

The orthograde labeling of postganglionic sympathetic cardiac nerves in the dog and cat as demonstrated by autoradiography

Previous functional and anatomical techniques have characterized the cardiac nerves and plexuses. They cannot, however, determine the course of fibers arising from a specific ganglion. This study has found that the intraaxonal orthograde labeling of axons can be used to determine the course of postganglionic sympathetic cardiac fibers in the dog and cat. The canine left caudal cervical ganglion and the feline right stellate ganglion were exposed through appropriate thoracotomies. Each ganglion received multiple injections of tritiated leucine (500 muCi/animal). Following a 3--14-day survival period the anesthetized animals were sacrificed by vascular perfusion. The injected ganglia, extracardiac nerves and selected portions of the heart were processed for autoradiography. Autoradiographs from the dog demonstrated labeled postganglionic sympathetic nerves in the extracardiac plexus, left atrial epicardium and the epicardium beneath the coronary sulcus. Labeled nerves in the cat heart were found within the epicardial layers and associated with blood vessels in the left ventricular myocardium. Neither myelinated fibers traveling through the injection site nor intrinsic cardiac ganglion cells were labeled, although the latter were often closely approximated to heavily labeled fibers.     

Transneuronal labeling of cochlear nucleus neurons by HRP-labeled auditory nerve fibers and olivocochlear branches in mice.

Auditory nerve fibers were labeled by extracellular injections of horseradish peroxidase into the spiral ganglion in mice. The labeled fibers were traced in an anterograde direction through the auditory nerve into the cochlear nucleus. In almost half of the injections, the labeled endings of auditory nerve fibers contacted cochlear nucleus neurons that were also labeled with horseradish peroxidase and were presumably transneuronally labeled. Only darkly labeled endings were associated with transneuronally labeled neurons, but not all darkly labeled endings had targets that were transneuronally labeled. Transneuronal labeling occurred almost exclusively in the ventral cochlear nucleus, often between endbulbs and bushy cells. Both "modified" endbulbs and the larger endbulbs of Held transneuronally labeled the bushy cells that they contacted. At the ultrastructural level, transneuronal labeling was evident as a darkening of ribosomes and the membrane surfaces of mitochondria, endoplasmic reticulum, and the nucleus. Transneuronal labeling occurred rarely in octopus, small, and stellate cells, and in neurons of the dorsal cochlear nucleus. Spiral ganglion injections also label olivocochlear fibers, efferent fibers that pass through the ganglion en route to the hair cells. These fibers give off branches to the cochlear nucleus that were rarely associated with transneuronal labeling. In eight instances, the targets of olivocochlear branches were stellate cells or small cells. We suggest that in our mouse preparation, horseradish peroxidase is effective as a transneuronal marker because the short distance from injection site to the cochlear nucleus results in a high concentration of horseradish peroxidase in the endings of the auditory nerve fibers.     

Localization of sensory neurons traversing the stellate ganglion of the cat

The distribution of sensory cells whose axons traverse the stellate ganglion and project via sympathetic cardiac nerves to the heart of the cat has been examined quantitatively. Horseradish peroxidase (HRP) injected at multiple sites in the right stellate ganglion, or applied to the middle cardiac nerve, labelled small numbers of cells in the thoracic dorsal root ganglia (DRG) from T₁ to T₈. These cells were most numerous between T₂ and T₅ and were consistently small (< 40 μm) relative to other cells in the DRG. When HRP was applied to middle cardiac nerves, the numbers of labelled sensory cells always exceeded the numbers of myelinated axons counted in the same nerves from other cats. It is concluded that the distribution of the cells of cardiac sensory fibres is more extensive within thoracic DRG than has been previously reported, and it is suggested that such fibres travelling in the sympathetic cardiac nerves may be either myelinated or unmyelinated.     

Medullary cells of origin of physiologically identified cardiac nerves in the dog

Individual cardiac nerves from which stimulation elicited cardioinhibition (bradycardia and negative inotropism) were identified in 24 of 38 dogs. Subsequently, 3-25 microliters of 30% horseradish peroxidase (HRP) were injected into an identified cardiac nerve. After a 3-day survival period, the medulla oblongata was processed for HRP histochemistry. Retrograde labeling was observed to be concentrated primarily in the ipsilateral nucleus ambiguus (NA) and in medium-sized neurons located ventral and lateral to the larger neurons of the principal NA cell column. This latter location was so characteristic that it has been designated the ventrolateral nucleus ambiguus (VLNA). Labeled neurons were found at all levels of the NA and VLNA and their distribution was similar irrespective of the cardiac nerve injected. Relatively few labeled neurons were observed in the dorsal motor nucleus of the vagus nerve (DMV) except after injections into the left and right recurrent cardiac nerves and the left cranial vagal nerve. In some dogs labeled cells were present only in and ventrolateral to the NA and not in the DMV, even though stimulation of the injected nerve elicited both bradycardia and negative inotropism. These results demonstrate that ventrolateral regions of the NA represent the major site of cardioinhibitory motor neurons in the dog that they can regulate both rate and force.     

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.     

The ovarian innervation in the dog: a preliminary study for the base for electro-acupuncture.

The origin of the canine ovarian sensory and sympathetic nerves was studied by applying horseradish peroxidase (HRP) or wheat germ agglutinin conjugated to HRP (WGA-HRP) to the ovarian stroma and into the ovarian bursa. HRP/WGA-HRP positive neurons were found bilaterally in the dorsal root ganglia of T10 to L4 segment with the majority located in T13 to L2. In sympathetic paravertebral ganglia, labeled neurons were distributed bilaterally in ganglia from T11 to L4 with the majorities located in segments T13 to L2. Both distributions show ipsilateral predominance. Labeled prevertebral neurons were mainly located in the aorticorenal ganglion, ovarian ganglia and caudal mesenteric ganglion. No labeled neurons were found in the dorsal motor nucleus of vagus, nodose ganglia or sacral segment from S1 to S3. This study provides the possible morphological basis of electro-acupuncture concerning the somato-visceral reflex of the ovary.     

Organization of the sympathetic postganglionic innervation of the rat heart

The origins and organization of cardiac sympathetic postganglionic nerves in the rat were identified in the present investigation. The retrograde tracer, Diamidino Yellow, was injected into the right or left ventricles to label somata in the sympathetic chain. Analysis of all sympathetic ganglia from superior cervical ganglion through the 10th thoracic ganglion indicated that the postganglionic innervation of the rat cardiac ventricles originates bilaterally. The majority of these somata were located in the middle and inferior cervical ganglia (middle cervical-stellate ganglion complex) (approximately 92% of all labelled cells), with lesser contributions from the superior cervical and 4th through 6th thoracic ganglia. To confirm and further quantitate these findings, the middle cervical-stellate ganglion complex was removed (MC-S ganglionectomy) bilaterally or ipsilaterally from the left or right sides, and regional cardiac norepinephrine concentration (left and right atrial appendages and left and right ventricles) was analysed 7 or 28 days later. At both times after bilateral MC-S ganglionectomy, regional cardiac norepinephrine was reduced by 89% to 100%, indicating the removal of almost all cardiac noradrenergic cells of origin and possibly fibers of passage. The results of unilateral MC-S ganglionectomy experiments indicated that the atrial appendages and the left ventricle receive bilateral innervation from the middle cervical-stellate ganglion complex. However, the left middle cervical-stellate ganglion complex appears to contribute a majority of the norepinephrine to the right ventricle. Furthermore, between 7 and 28 days after contralateral MC-S ganglionectomy, atrial appendages, but not ventricles, display significant recovery of norepinephrine content. The present data demonstrate: (1) a bilateral locus of origin of cardiac sympathetic postganglionic neurons, limited longitudinally to cervical through mid-thoracic ganglia, and (2) the ability of the cardiac postganglionic innervation to regenerate after partial denervation. These results demonstrate anatomical evidence for significant bilateral integration of cardiac sympathetic activity at the level of the sympathetic ganglion in the rat.     

Sympathetic and sensory neurons projecting into the cervical sympathetic trunk in the chicken.

The cell bodies of the sensory and sympathetic pre- and postganglionic neurons projecting into the cervical sympathetic trunk were retrogradely labeled with horseradish peroxidase in the chicken. Preganglionic neurons were located in the spinal segments T1-T6 (maximum T2), postganglionic neurons in the paravertebral ganglia T1-T3 (maximum T1) and sensory neurons in the dorsal root ganglia T1-T4 (maximum T1). Labeled preganglionic neurons were widely distributed across the intermediate gray matter and lateral funiculus, but the majority of them were located in the intermediomedial area dorsolateral to the central canal. The short and long axis diameters of labeled preganglionic neurons in this area decreased caudally. From the data of the present study, it is estimated that about 4190 preganglionic, about 450 postganglionic and about 390 sensory neurons project into the cervical sympathetic trunk cranial to the paravertebral ganglion T1 in the chicken.     

The superior cervical ganglion: Origin of sympathetic fibers in the facial and hypoglossal nerves in the cat

Location of superior cervical ganglion (SCG) neurons, sending axons into the facial and hypoglossal nerves, was investigated in the cat by means of retrograde axonal transport of horseradish peroxidase (HRP). After wheat germ agglutinin conjugated HRP (WGA-HRP) was injected into these nerves, many retrogradely labeled neurons were found widely in the ipsilateral SCG, particularly around the caudal half of the SCG. These neurons were round or oval in shape and 70-80% of these were medium in size. In fluorescent experiments, fast blue (FB) was used in combination with diamidino yellow (DY). After injections of FB into the facial nerve and DY into the hypoglossal nerve, a few FB-DY double-labeled neurons occurred in the SCG ipsilaterally.