Innervation of the C cells of chicken ultimobranchial glands studied by immunohistochemistry, fluorescence microscopy, and electron microscopy

Innervation of the ultimobranchial glands in the chicken was investigated by immunohistochemistry, fluorescence microscopy and electron microscopy. The nerve fibers distributed in ultimobranchial glands were clearly visualized by immunoperoxidase staining with antiserum to neurofilament triplet proteins (200K-, 150K- and 68K-dalton) extracted from chicken peripheral nerves. The ultimobranchial glands received numerous nerve fibers originating from both the recurrent laryngeal nerves and direct vagal branches. The left and right sides of the ultimobranchial region were asymmetrical. The left ultimobranchial gland had intimate contact with the vagus nerve trunk, especially with the distal vagal ganglion, but was somewhat separated from the recurrent nerve. The right gland touched the recurrent nerve, the medial edge being frequently penetrated by the nerve, but the gland was separated from the vagal trunk. The left gland was innervated mainly by the branches from the distal vagal ganglion, whereas the right gland received mostly the branches from the recurrent nerve. The carotid body was located cranially near to the ultimobranchial gland. Large nerve bundles in the ultimobranchial gland ran toward and entered into the carotid body. By fluorescence microscopy, nerve fibers in ultimobranchial glands were observed associated with blood vessels. Only a few fluorescent nerve fibers were present in close proximity to C cell groups; the C cells of ultimobranchial glands may receive very few adrenergic sympathetic fibers. By electron microscopy, numerous axons ensheathed with Schwann cell cytoplasm were in close contact with the surfaces of C cells. In addition, naked axons regarded as axon terminals or "en passant" synapses came into direct contact with C cells. The morphology of these axon terminals and synaptic endings suggest that ultimobranchial C cells of chickens are supplied mainly with cholinergic efferent type fibers. In the region where large nerve bundles and complex ramifications of nerve fibers were present, Schwann cell perikarya investing the axons were closely juxtaposed with C cells; long cytoplasmic processes of Schwann cells encompassed large portions of the cell surface. All of these features suggest that C-cell activity, i.e., secretion of hormones and catecholamines, may be regulated by nerve stimuli.     

Mash1 is required for glomus cell formation in the mouse carotid body

The carotid body consists of chemoreceptive glomus cells, sustentacular cells and nerve endings. The murine carotid body, located at the carotid bifurcation, is always joined to the superior cervical ganglion of the sympathetic trunk. Glomus cells and sympathetic neurons are immunoreactive for the TuJ1, PGP9.5, tyrosine hydroxylase (TH) and neuropeptide Y (NPY) markers. Glomus cells are also immunoreactive for serotonin (5-HT). A targeted mutation of Mash1, a mouse homolog of the Drosophila achaete-scute complex, results in the elimination of sympathetic ganglia. In Mash1 null mutant mice, the carotid body primordium forms normally in the wall of the third arch artery at embryonic day (E) 13.0 and continues to develop, although the superior cervical ganglion is completely absent. However, no cells in the mutant carotid body display the TuJ1, PGP 9.5, TH, NPY and 5-HT markers throughout development. The absence of glomus cells was also confirmed by electron microscopy. The carotid body of newborn null mutants is composed of mesenchymal-like cells and nerve fibers. Many cells immunoreactive for the S-100 protein, a sustentacular cell marker, appear in the mutant carotid body during fetal development. The Mash1 gene is thus required for the genesis of glomus cells but not for sustentacular cells.     

The sources of the innervation of the sphincter of the esophagogastric junction in rats]

The investigation has been performed by means of the luminescent microscopical method. The retrograde axonal transport of the fluorescent marker primuline has demonstrated that a definite amount of labelled cells are observed in the celiac plexus, in nodes of the thoracic part of the sympathetic trunk (predominantly in Th6-Th8). Innervation of the EGP sphincter is mainly performed from the sympathetic trunk nodes (Th6-Th8) and from the celiac plexus.     

VIP-, galanin-, and neuropeptide-Y-immunoreactive fibers in the chicken carotid bodies after various types of denervation

The chicken carotid body receives numerous branches from the vagus nerve, especially distal (nodose) ganglion, and the recurrent laryngeal nerve. Dense networks of peptidergic nerve fibers immunoreactive for substance P, calcitonin gene-related peptide (CGRP), galanin, vasoactive intestinal peptide (VIP) and neuropeptide Y are distributed in and around the carotid body. Substance-P- and CGRP-immunoreactive fibers projecting to the chicken carotid body mainly come from the vagal ganglia. In the present study, various types of denervation experiments were performed in order to clarify the origins of VIP-, galanin- and neuropeptide-Y-immunoreactive fibers in the chicken carotid bodies. After nodose ganglionectomy, midcervical vagotomy or excision of the recurrent laryngeal nerve, VIP-, galanin- and neuropeptide-Y-immunoreactive fibers were unchanged in the carotid body region. Furthermore, these peptidergic fibers remained unaffected even by removal of the nodose ganglion in conjunction with severance of the recurrent laryngeal nerve that induced a marked decrease in TuJ1-immunoreactive fibers in the carotid body region. VIP-, galanin- and neuropeptide-Y-immunoreactive fibers are densely distributed around the arteries supplying the carotid body in normal chickens. The peptidergic fibers around the arteries were also unaffected after the denervation experiments. However, after removal of the 14th cervical ganglion of the sympathetic trunk, which lies close to the vertebral artery on the root of the brachial plexus and issues prominent branches to the artery, VIP-, galanin- and neuropeptide-Y-immunoreactive fibers almost disappeared in the carotid body region. The ganglion contained many VIP-, galanin- and neuropeptide-Y-immunoreactive neurons. Thus it is clear that VIP-, galanin- and neuropeptide-Y-immunoreactive fibers in the chicken carotid body region are mainly derived from the 14th cervical sympathetic ganglion via the vertebral artery.     

Sympathetic innervation of the pulmonary artery,ascending aorta,and coronar glomera of the rabbit

Summary Adrenergic nerve fibres were demonstrated in the connective tissue of the rabbit coronar glomera by means of the formaldehyde-induced fluorescence technique for catecholamines. This type of innervation is similar to the adrenergic nerve supply to the rabbit and cat carotid body. Adrenergic fibres terminate subendothelially and only a few can be traced to type I cells in the glomera coronaria. The sympathetic innervation of the ascending aorta is exceedingly sparse in contrast to the pulmonary trunk, while vasa vasorum of the ascending aorta exhibit a dense sympathetic innervation.     

Heterogeneous expression of TASK-3 and TRAAK in rat paraganglionic cells

In the present study, we investigated the immunohistochemical localization of the two-pore K⁺ channels, TASK-3 and TRAAK, in paraganglionic cells within the superior cervical ganglion, stellate ganglion, and aortic body in comparison with membrane channels in chief cells of the carotid body. TASK-3 immunoreactivity was observed in the paraganglionic cells in all tissues examined. TRAAK immunoreactivity was observed in the chief cells of the aortic body as well as these of the carotid body, but not in the paraganglionic cells in the sympathetic (superior cervical and stellate) ganglia. Our findings indicate that sympathetic paraganglionic cells and glossopharyngeal/vagal paraganglionic cells were different from each other in the expression patterns of TASK-3 and TRAAK to result in the different chemoreception properties of sympathetic paraganglionic cells from those of chief cells of the aortic and carotid bodies.     

Evidence for GABAergic fibers entering the superior cervical ganglion of rat from the preganglionic nerve trunk

Summary The origin of gamma-aminobutyric acid immunoreactive (GABA-IR) nerve fibers present in the superior cervical ganglion (SCG) of rat was investigated. With immunocytochemical techniques many nerve fibers showed GABA-like positivity in the cervical sympathetic trunk, whereas similar staining could not be revealed in the internal carotid nerve or in the external carotid nerve. Ligation of the cervical sympathetic trunk for 24 h resulted a dramatic reduction in the staining density in the ganglion and in the cervical sympathetic trunk distal to the ligature. After transection of the preganglionic nerve fibers for eleven days or more, very few fibers staining for GABA were seen in the ganglion. The immunohistochemical results suggest that a major source of GABA within the SCG is a population of GABAergic axons entering from the preganglionic trunk.     

EFFECTS OF SINUS NERVE STIMULATION ON CAROTID BODY GLOMUS CELLS

The sinus nerve or sympathetic trunk was stimulated unilaterally in one group of adult cats or Syrian hamsters while in another group the sinus nerve or sympathetic trunk was cut unilaterally and the animals were given reserpine. In a third group, atropine was administered prior to sinus nerve stimulation. All tissues were processed for the detection of primary monoamines. The carotid bodies on the operated sides were compared with those on the unoperated sides of the same animal in order to determine if amine depletion occurred following the experimental procedures. After sinus nerve stimulation alone, the density of the granules in the glomus cells was decreased, but changes were not noted in the granules following sympathetic nerve stimulation. Sinus nerve stimulation after atropine administration resulted in no change in granule density. Sinus nerve transection followed by reserpine treatment resulted in a greater decrease in granule density on the unoperated than on the operated side. Transection of the sympathetic components to the carotid body followed by reserpine injections resulted in a decrease in granule density in the glomus cells on both the operated and unoperated sides. These results suggest that the sinus nerve must be intact for reserpine to exert an effect and that the sinus nerve may contain efferent fibers which modulate amine secretion.     

Evidence for GABAergic fibers entering the superior cervical ganglion of rat from the preganglionic nerve trunk

The origin of gamma-aminobutyric acid immunoreactive (GABA-IR) nerve fibers present in the superior cervical ganglion (SCG) of rat was investigated. With immunocytochemical techniques many nerve fibers showed GABA-like positivity in the cervical sympathetic trunk, whereas similar staining could not be revealed in the internal carotid nerve or in the external carotid nerve. Ligation of the cervical sympathetic trunk for 24 h resulted a dramatic reduction in the staining density in the ganglion and in the cervical sympathetic trunk distal to the ligature. After transection of the preganglionic nerve fibers for eleven days or more, very few fibers staining for GABA were seen in the ganglion. The immunohistochemical results suggest that a major source of GABA within the SCG is a population of GABAergic axons entering from the preganglionic trunk.     

FRS2 alpha 2F/2F mice lack carotid body and exhibit abnormalities of the superior cervical sympathetic ganglion and carotid sinus nerve

The docking protein FRS2α is an important mediator of fibroblast growth factor (FGF)-induced signal transduction, and functions by linking FGF receptors (FGFRs) to a variety of intracellular signaling pathways. We show that the carotid body is absent in FRS2α^2F/2F mice, in which the Shp2-binding sites of FRS2α are disrupted. We also show that the carotid body rudiment is not formed in the wall of the third arch artery in mutant embryos. In wild-type mice, the superior cervical ganglion of the sympathetic trunk connects to the carotid body in the carotid bifurcation region, and extends thick nerve bundles into the carotid body. In FRS2α^2F/2F mice, the superior cervical ganglion was present in the lower cervical region as an elongated feature, but failed to undergo cranio-ventral migration. In addition, few neuronal processes extended from the ganglion into the carotid bifurcation region. The number of carotid sinus nerve fibers that reached the carotid bifurcation region was markedly decreased, and baroreceptor fibers belonging to the glossopharyngeal nerve were absent from the basal part of the internal carotid artery in FRS2α^2F/2F mutant mice. In some of the mutant mice (5 out of 14), baroreceptors and some glomus cells were distributed in the wall of the common carotid artery, onto which the sympathetic ganglion abutted. We propose that the sympathetic ganglion provides glomus cell precursors into the third arch artery derivative in the presence of sensory fibers of the glossopharyngeal nerve.     

KISS1 and KISS1R expression in the human and rat carotid body and superior cervical ganglion

KISS1 and its receptor, KISS1R, have both been found to be expressed in central nervous system, but few data are present in the literature about their distribution in peripheral nervous structures. Thus, the aim of the present study was to investigate, through immunohistochemistry, the expression and distribution of KISS1 and KISS1R in the rat and human carotid bodies and superior cervical ganglia, also with particular reference to the different cellular populations. Materials consisted of carotid bodies and superior cervical ganglia were obtained at autopsy from 10 adult subjects and sampled from 10 adult Sprague-Dawley rats. Immunohistochemistry revealed diffuse expression of KISS1 and KISS1R in type I cells of both human and rat carotid bodies, whereas type II cells were negative. In both human and rat superior cervical ganglia positive anti-KISS1 and -KISS1R immunostainings were also selectively found in ganglion cells, satellite cells being negative. Endothelial cells also showed moderate immunostaining for both KISS1 and KISS1R. The expression of both kisspeptins and kisspeptin receptors in glomic type I cells and sympathetic ganglion cells supports a modulatory role of KISS1 on peripheral chemoreception and sympathetic function. Moreover, local changes in blood flow have been considered to be involved in carotid body chemoreceptor discharge and kisspeptins and kisspeptin receptors have also been found in the endothelial cells. As a consequence, a possible role of kisspeptins in the regulation of carotid body blood flow and, indirectly, in chemoreceptor discharge may also be hypothesized.     

Blood supply of the human cervical sympathetic chain and ganglia

OBJECTIVE: Cadaveric studies of the blood supply to the human cervical sympathetic chain and ganglia are lacking in the English literature. This study seeks to elucidate the gross blood supply of the cervical sympathetic chain so as to avoid surgical disruption of these vessels and thus decrease the risk of vascular insufficieny and subsequent dysfunction of thoracolumbar autonomic outflow to the head and neck. METHODS: Twelve (24 sides) human cadavers (8 male and 4 female) were dissected and their brachiocephalic veins, internal carotid arteries, and vertebral arteries cannulated. Red and blue latex was injected into the arteries and veins respectively. Dissection of the neck was carefully performed and the blood supply of the cervical sympathetic chain identified. RESULTS: The primary arterial supply to the sympathetic chain and ganglia were from superior to inferior the ascending pharyngeal, ascending cervical, thyrocervical trunk, and supreme intercostal arteries. The primary venous drainage of these structures was primarily by direct posterior branches into the internal jugular vein. In addition, we have found an area at the junction of the lower two-thirds and upper one-third of the neck, which is deficient in blood supply (both arterial and venous). CONCLUSIONS: Although sympathetic injury is a rare consequence of cervical operations, the current data should be useful to the surgeon who operates in the cervical region so as to avoid potential complications from disruption of the primary blood supply of the cervical sympathetic chain and ganglia. Also, future techniques of selective iatrogenic disruption of the blood supply to portions of these structures e.g. stellate ganglion may be helpful in treating entities such as hyperhydrosis.     

Age-related changes in the amount and intensity of the fluorescence of small, intensely fluorescing cells in the autonomic ganglia of rats

The number and intensity of fluorescence of small, intensely fluorescent cells were measured on serial slices of main pelvic (MPG) ganglion and lumbar ganglia of sympathetic trunk (LG), treated by modified Falck method, on days 1, 7, 14, 28, and 26-30 months of age. The content of paraform-induced fluorescence increased with age of two weeks and later in SIF cells of MPG and LG. The number of SIF cells in LG decreased with age, while that of MPG increased. The growth of a number of SIF cells in MPG was detected in large clusters.     

Development of the sympathetic system in the Mexican axolotl, Ambystoma mexicanum

Migration of trunk neural crest cells in axolotl embryos has been followed by autoradiography using grafts of [³H]thymidine-labeled neural folds. Crest cells form melanocytes, dorsal fin mesenchymal cells, spinal ganglion cells, and reach the sympathetic region. Sympathetic neurons, however, are not identifiable morphologically until about 6 weeks posthatching, in 24-mm larvae. At this stage, neurons, although few, have characteristic ultrastructure and receive synapses. The diffuse ganglia also contain innervated chromaffin cells; these differentiate earlier, a few days posthatching, in 14-mm larvae. A third type of cell is of morphologically indifferent appearance. Catecholamine-specific formaldehyde-induced fluorescence first appears clearly at 14 mm; with growth, the number of fluorescent cells increases. Series of larvae were injected intraperitoneally with nerve growth factor (NGF), six 30-unit injections over 2 weeks. NGF treatment increases the number of neurons apparent in 24-mm larvae. Furthermore, differentiated neurons occur in NGF-treated 20-mm larvae (about 4 weeks posthatching), when there are none in controls. The early appearance of differentiated chromaffin cells and the relatively late appearance of differentiated sympathetic neurons suggest that adrenergic functions during the first few weeks of larval life are controlled humorally by the chromaffin cells, and that at 24 mm, neurons begin to provide faster, finer control.     

Sympathetic innervation of the carotid bifurcation in the rabbit and cat: Blood vessels,carotid body and carotid sinus

Summary Two postganglionic branches of the superior cervical ganglion enter the area of the carotid bifurcation in the rabbit and the cat. The common and external carotid arteries receive a rich adrenergic nerve supply, which can be demonstrated by fluorophores of biogenic amines appearing after formaldehyde treatment. The internal carotid artery is only sparsely innervated; however, it shows a dense sympathetic supply at the site of pressor receptors. Following removal of the superior cervical ganglion, a total loss of fluorescent adrenergic nerves occurs and degeneration of nerve endings possessing dense core vesicles is conspicuous. These nerve terminals are situated mainly subendothelially in the carotid body sinusoids; they only rarely terminate on type I cells.     

Immunohistochemical and histochemical evidence for the presence of noradrenaline,serotonin and gamma-aminobutyric acid in chief cells of the mouse carotid body

The immunohistochemical study revealed tyrosine hydroxylase (TH), dopamine -hydroxylase (DBH), phenylethanolamine N-methyltransferase (PNMT), serotonin, glutamate decarboxylase (GAD) and -aminobutyric acid (GABA) immunoreactivities in the mouse carotid body. TH and DBH immunoreactivities were found in almost all chief cells and a few ganglion cells, and in relatively numerous varicose nerve fibers of the carotid body. The histofluorescence microscopy showed catecholamine fluorescence in almost all chief cells. However, no PNMT immunoreactivity was observed in the carotid body. Serotonin, GAD and GABA immunoreactivities were also seen in almost all chief cells of the carotid body. From combined immunohistochemistry and fluorescence histochemistry, catecholamine and serotonin or catecholamine and GABA were colocalized in almost all chief cells. Thus, these findings suggest that noradrenaline, serotonin and GABA may be synthesized and co-exist in almost all chief cells of the mouse carotid body and may play roles in chemoreceptive functions.     

Histochemical observations on cholinesterase activity in the autonomic ganglia of the human sympathetic trunk and vagus nerve

Synopsis The distribution of cholinesterase activity was studied histochemically in the autonomic ganglia of the human sympathetic trunk and the vagus nerve using a modified Koelle's technique. It was found that the cytoplasm of both sympathetic and parasympathetic nerve cells contained acetylcholinesterase but the intensity of the enzyme reaction varied from cell to cell in both types of ganglia. Tissue elements surrounding the nerve cells showed a low butyrylcholinesterase activity in the ganglia of the sympathetic trunk but a high one in the terminal ganglia of the vagus nerve. Postganglionic nerves fibres gave a weak reaction for acetylcholinesterase in the sympathetic, but a strong one in the vagus ganglia. The distribution pattern of cholinesterases in human autonomic ganglia was found to be different from that of a variety of laboratory and wild animals.     

Histochemically demonstrable catecholamines in sympathetic ganglia and carotid body of spontaneously hypertensive and normotensive rats

The catecholamine content and morphology of the superior cervical and the hypogastric ganglion and the carotid body were studied in Spontaneously Hypertensive Rats (SHR) before (at the age of 6 weeks) and after (at the age of 20 weeks) becoming hypertensive, with Wistar Kyoto (WKY) rats as controls. The study was performed by formaldehyde-induced fluorescence method combined with quantitative microfluorimetry of catecholamines. At the age of 6 weeks the only significant difference observed between the rat strains was a greater number of small intensely fluorescent (SIF) cells in the superior cervical ganglion of SHR. At the age of 20 weeks the fluorescence intensity was higher in the principal neurons of the superior cervical ganglion and in glomus cells of the carotid body of SHR compared to WKY. The volumes of superior cervical ganglion and carotid body were larger in 20-week-old SHR compared to WKY. In the hypogastric ganglion differences were not found between SHR and WKY rats. The present results show differences in the superior cervical ganglion and in the carotid body of adult SHR compared to controls. These differences develop during the time period when the SHR become hypertensive, and might be functionally significant in the regulation or maintenance of the increased blood pressure in SHR rats.     

Histofluorescence and ultrastructural observations of small intensely fluorescent (SIF) cells in the superior sympathetic ganglion of the guinea pig

Summary Amine-containing small intensely fluorescent (SIF) cells are ubiquitous in vertebrate sympathetic ganglia and, in some species, SIF cells have been identified as interneurons. The hypothesis proposed in this study is that SIF cells in superior sympathetic ganglia of the guinea pig function as interneurons, with efferent connections characteristic for the species. Fluorescence (catecholamine) microscopy and 5-hydroxydopamine marker for electron microscopy were used to study SIF cells, their processes and connections in this ganglion.Brightly fluorescent fibers were seen attached to virtually all SIF cells, and were of two types. The first type, single or arranged in cords, interconnected elements of the SIF-cell system; these apparent linkages joined individual SIF cells as well as adjacent clusters. The electron-microscopic evidence for synaptic contacts between SIF cells warrants the claim that integrated action is a presumed function of these elements. The second type of SIF-cell process was generally of greater length. These individual, branching fibers made presumed connections with dendrites of most principal ganglionic neurons. This arrangement suggested by histofluorescence preparations was confirmed by electron microscopy to involve synaptic connections, and the postsynaptic element was shown to be continuous with the perikaryon of the principal ganglionic neuron. Ultrastructural evidence that collections of dense-cored vesicles occur within processes of both principal ganglionic neurons and SIF cells, in proximity to unsheathed portions of plasma membrane, leads to the conclusion that interstitial diffusion of catecholamine from both may occur; the finding of SIF cell processes adjacent to fenestrated blood vessels suggests that catecholamine may also be transported through capillaries.