Substance P as a baro- and chemoreceptor afferent neurotransmitter: immunocytochemical and neurochemical evidence in the rat

The possibility that substances P (SP) is a neurotransmitter of baro- and chemoreceptor afferents in the rat was investigated. SP-like immunoreactivity (SP-I) was analyzed quantitatively by radioimmunoassay in various levels of the nucleus tractus solitarius (NTS), the site of termination of these afferents while SP-containing afferent neurons were studied in various portions of the peripheral pathways by immunocytochemistry. It was found that the NTS contained significant amounts of SP-I and that unilateral removal of the nodose ganglia reduces the SP-I content of those portions of the NTS known to receive vagal afferents. In addition, SP-I was visualized in discrete fibers in the tunica adventitia of the aortic arch and carotid sinus regions, the vagus nerve and nodose ganglia. These results in the rat are consistent with our previous studies in the cat and provide further evidence that SP is contained within baro- and chemoreceptor afferent nerves.     

Ultrastructural changes in the rat carotid body in severe haemorrhagia

Summary Rat carotid bodies were studied electron microscopically after short-term severe hypovolaemia, which is known to induce a marked chemoreceptor activation in the carotid body. Altogether 84 nerve-endings in the hypovolaemic rats' carotid bodies and 91 nerve-endings in the control carotid bodies were investigated. An increased accumulation of the glomus cell granular vesicles near the synaptic specializations of the nerve-endings was observed after hypovolaemia. Moreover, a statistically significant increase in the contacts between the nerve-ending synaptic specializations and the glomus cell granular vesicles was observed after hypovolaemia. A suggestion was made that the glomus cells might act as modulating, probably inhibitory, interneurones, whose catecholamines are responsible for the inhibition.The authors are greatly indebted to lecturer Pekka Korkala Ph.L. from the Department of Psychology for his skilful statistical analysis of the results.     

Calbindin D-28k immunoreactive nerve fibers in the carotid body of normoxic and chronically hypoxic rats

The distribution and ultrastructural characteristics of calbindin D-28k immunoreactive nerve fibers were examined in the carotid body of the normoxic control rats by light and electron microscopy, and the abundance of calbindin D-28k fibers in the carotid body was compared in normoxic and chronically hypoxic rats (10% O2 and 3.0-4.0% CO2 for 3 months). Calbindin D-28k immunoreactivity was recognized in nerve fibers within the carotid body. Calbindin D-28k immunoreactive nerve fibers appeared as thin processes with many varicosities. They were distributed around clusters of glomus cells, and around blood vessels. Immunoelectron microscopy revealed that the calbindin D-28k immunoreactive nerve terminals are in close apposition with the glomus cells, and membrane specialization is visible in some terminals. Some dense-cored vesicles in the glomus cells were aggregated in this contact region. The chronically hypoxic carotid bodies were found to be enlarged several fold, and a relative abundance of calbindin D-28k fibers was lesser than in the normoxic carotid bodies. When expressed by the density of varicosities per unit area of the parenchyma, the density of calbindin D-28k fibers associated with the glomus cells in chronically hypoxic carotid bodies was decreased by 70%. These immunohistochemical findings indicate a morphological basis for involvement of calcium binding protein in the neural pathway that modulates carotid body chemoreception.     

Regulation of chemoreceptor sensitivity in the carotid body: the role of presynaptic sensory nerves

Several neural and vascular mechanisms regulate the sensitivity of carotid body chemoreceptors to hypoxia, hypercapnia, and acidosis. Factors that control blood flow and oxygen delivery in the carotid body along with those that augment or diminish catecholamine release from glomus cells can have major effects on chemoreceptor function. In addition, the sensory nerves themselves may participate in the regulation of chemoreceptor sensitivity. A portion of the carotid body's sensory nerves are presynaptic to glomus cells. In response to stimulation, the sensory nerve terminals exhibit ultrastructural changes that resemble changes associated with increased release of transmitter from motor nerves: 1) the number of small (synaptic) vesicles decreases; and 2) coated vesicles and coated regions of cisternal membrane increase in number during stimulation. If sensory nerves of the carotid body release a neurotransmitters, sensory nerve activity could influence glomus cell secretion of catecholamines or other substances tha modify chemoreceptor sensitivity. Such an effect could be produced in the carotid body by hypoxia and other conditions that stimulate the sensory nerves or it could result from antidromic activity evoked in the sensory nerves by primary afferent depolarization of their terminals in the CNS.     

Ontogeny of the carotid body and glomus cells distributed in the wall of the common carotid artery and its branches in the chicken

Summary Developmental patterns of immunoreactivity for serotonin and neuropeptide Y were investigated immunohistochemically in the carotid body and glomus cells in the wall of the common carotid artery and around its branches of chickens at various developmental ages. The development of peptidergic nerve fibers was also studied. Serotonin immunoreactivity began to appear in the glomus cells of the carotid body and around arteries at 10 days of incubation and became very intense from 12 days onwards. Neuropeptide Y immunoreactivity also appeared in these cells at 10 days, became intense at 14 days, and was sustained until 20 days. After hatching, neuropeptide Y immunoreactivity in the carotid body rapidly decreased with age and almost cisappeared at posnatal day 10. However, it persisted for life in the glomus cells distributed in the wall of the common carotid artery. Substance P- and calcitonin gene-related peptide (CGRP)-immunoreactive fibers first penetrated into the carotid body parenchyma at 12 days of incubation. These peptidergic nerve fibers in the carotid body and glomus cell groups in and around arteries gradually increased with age, and approached the adult state at 18 days of incubation. Only a few galanin-and vasoactive intestinal peptide (VIP)-immunoreactive fibers were observed in the late embryonic carotid bodies. They rapidly developed after hatching and reached adult numbers at postnatal day 10. During late embryonic and neonatal development, considerable numbers of met-enkephalin-immunoreactive fibers were detected in the connective tissue encircling the carotid body.     

Dense granule-containing cells in arterial chemoreceptor areas of the tortoise (Testudo hermanni)

Light- and electron-microscopic observations of the chemosensory areas of the arteries of the tortoise (Testudo hermanni) reveal that clusters of nonmuscular cells are found in the adventitial layer of restricted regions of the carotid artery, aortic arch, and pulmonary artery. In these clusters, three types of cells are complexly interwoven: the G-cell closely resembles the glomus cell, which has been found in the arterial chemoreceptor area of several animal species; the LG-cell has very large electron-dense granules; the third type is a G- and LG-cell supporting cell. Membrane specializations are often observed at apposing membranes between G-cells. Two kinds of nerve endings synapse with G-cells, one with numerous clear synaptic vesicles, the other without vesicles. Some G-cells are in membrane-to-membrane contact with smooth-muscle cells (g-s connection), and here a membrane thickening is visible. Nerve terminals with numerous synaptic vesicles synapse with the LG-cells. The G-cell in the carotid artery, the aorta, and the pulmonary artery is a chemoreceptor element ultrastructurally the same as the glomus cell in the arterial chemoreceptor area of various vertebrate species.     

Postnatal maturation of neuroepithelial bodies and carotid body innervation: A quantitative investigation in the rabbit

The intrapulmonary airways contain oxygen-sensitive chemoreceptors which may be analogous to the arterial chemoreceptors: the neuroepithelial bodies (NEB). While the NEB are prominent in the neonatal lung, physiological studies indicate that the carotid bodies are still relatively inactive at birth. This points to an unequal degree of development of both during the early neonatal period. As a reflexogenic chemoreceptor function depends on a well-developed innervation, we undertook a comparative investigation of the development of the NEB and the carotid body glomus cell innervation. Two morphological aspects of the innervation of NEB and carotid body glomus cells were quantified in rabbits of different age groups. The total sectional area of intracorpuscular and intraglomerular nerve endings per NEB or glomus cell group, respectively, was measured and the area percentage of mitochondria and synaptic vesicles was determined. In the NEB, no significant difference in total sectional area of the nerve endings between the age groups was observed, while in the carotid body there was a significant increase in the adult age group. In addition, the area percentage of mitochondria and synaptic vesicles of the nerve endings did not change significantly with age in the NEB, while in the carotid body these increased and decreased, respectively, with age. These observations point to a shift from morphologically efferent nerve endings, rich in synaptic vesicles, to morphologically afferent nerve endings, rich in mitochondria. Our interpretation of these findings is that, at birth, the NEB innervation is more mature than the carotid body glomus cell innervation and that the latter matures at a later time than the former. These findings support the theory that the NEB may act as complementary chemoreceptors to the carotid body during the early postnatal period.     

Immunocytochemical observations of corticotropin-releasing-factor-containing neurons in the rat hypothalamus with special reference to neuronal communication

Synapses between neurons with corticotropin-releasing-factor-(CRF)-like immunoreactivities and other immunonegative neurons in the hypothalamus of colchicine-treated rats, especially in the paraventricular nucleus (PVN) and the supraoptic nucleus (SON) were observed by immunocytochemistry using CRF antiserum. The immunoreactive nerve cell bodies and fibers were numerous in both the PVN and the SON. The CRF-containing neurons had synaptic contacts with immunonegative axon terminals containing a large number of clear synaptic vesicles alone or combined with a few dense-cored vesicles. We also found CRF-like immunoreactive axon terminals making synaptic contacts with other immunonegative neuronal cell bodies and fibers. And since some postsynaptic immunonegative neurons contained many large neurosecretory granules, they are considered to be magnocellular neurosecretory cells. These findings suggest that CRF functions as a neurotransmitter and/or modulator in addition to its function as a hormone.     

Glutamate in the glomus cells of the cat carotid body: Immunocytochemistry and in vitro release

The identity of the postulated excitatory transmitter released by glomus cells is not known. Since our preliminary work on paraffin sections of the cat carotid body indicated that most glomus cells were intensely immunoreactive to glutamate, we decided to investigate whether glutamate might be such a transmitter, using two approaches. One approach was to make a quantitative immunogold analysis of ultrathin sections to assess the level of glutamate immunoreactivity of glomus cells relative to glia and to afferent axon terminals. The other approach was to measure the potassium-induced release of glutamate from carotid bodies superfused in vitro. We consistently found that glomus cell profiles had 50% more immunogold particles per unit of area than glial cell or axonal profiles. However, the levels of glutamate immunoreactivity of glomus cells were lower than those expected for glutamatergic terminals. We also found that glutamate was not released from in vitro carotid bodies stimulated with high concentrations of potassium. These findings indicate that the oxygen-sensitive glomus cells have a high concentration of glutamate, which is not released by superfusion with high potassium. Thus, glutamate is not the excitatory transmitter released by glomus cells. We speculate that the high concentrations of glutamate might instead be related to the known dependence of the “in vitro” chemosensory activity on metabolic substrates.     

Ultrastructure of calcitonin gene-related peptide- and substance P-like immunoreactive nerve fibres in the carotid body and carotid sinus of the guinea pig

Previous studies have demonstrated that substance P- (SP) and calcitonin gene-related peptide-like immunoreactivities (CGRP-LI) coexist in sensory nerve fibres in the guinea-pig carotid body and carotid sinus. In the present study the ultrastructure of these nerve fibres was investigated by means of single- and double-labelling immunocytochemistry. In both, carotid body and carotid sinus immunoreactive fibres were unmyelinated axons of small diameter (0.12-0.56 microns). At the subcellular level, SP- and CGRP-LI were colocalized in intra-axonal dense core vesicles, suggesting corelease and simultaneous action of these two compounds. SP/CGRP-LI nerve fibres within the carotid body were mainly found in the interparenchymal connective tissue, but also occurred in relationship to blood vessels and nests of glomus cells. Neither in the carotid body not in the carotid sinus, SP/CGRP-LI axons corresponded to the large terminals which are generally considered to represent the main chemoreceptor and baroreceptor endings, respectively. Thus, SP/CGRP-LI fibres either belong to the chemo- and baroreceptors of the C-fibre class or constitute a fibre population not directly involved in conduction of baro- and chemoreflexes.     

Origin and colocalization of CGRP- and SP-reactive nerves in cat airway epithelium.

A combination of neuroanatomic techniques was used to examine the origin and neuropeptide content of nerve fibers in the airway epithelium of adult cats. By the use of immunocytochemical methods, the peptides substance P (SP) and calcitonin gene-related peptide (CGRP) were colocalized in airway epithelial nerve fibers. Two days after wheat germ agglutinin (WGA) was injected into the nodose ganglion, fibers containing WGA immunoreactivity (IR) were detected in the airway epithelium. SP-like immunoreactivity (LI) and CGRP-LI were demonstrated separately in the WGA-IR fibers, establishing their origin from nerve cell bodies of nodose ganglion. Vagal transection inferior to the nodose ganglion reduced the number of SP- and CGRP-IR fibers by greater than 90% in ipsilateral airways. In contralateral airways, SP-IR fibers were substantially reduced, whereas the effect on CGRP-IR fibers was not statistically significant. Vagotomy superior to the nodose ganglion did not alter the density of peptide-IR fibers. The results prove that SP- and CGRP-IR nerve fibers of cat airway epithelium originate from nerve cell bodies in the nodose ganglion and that SP- and CGRP-like peptides may be stored together in some nerve fibers of the airway epithelium.     

Heterogeneous distribution of neurocalcin-immunoreactive nerve terminals in the mouse adrenal medulla

Neurocalcin is a novel calcium-binding protein found in bovine brain tissue. We investigated immunoreactivity for neurocalcin in the mouse adrenal medulla using light and electron microscopy. The immunoreactivity was present in nerve fibers, nerve terminals, and ganglion cells in the adrenal medulla, but chromaffin cells, sustentacular cells, and Schwann cells were negative in reaction. Nerve bundles containing neurocalcin-immunoreactive fibers passed through the adrenal cortex and extended into the medulla. Immunopositive nerve fibers branched off and projected varicose terminals around the chromaffin cells. These varicose terminals contained small and large-cored vesicles and made synapses with the chromaffin cells. We performed paraformaldehyde-induced fluorescence-histochemical studies for catecholamine combined with immunohistochemical studies for neurocalcin. Neurocalcin-immunoreactive nerve terminals were more abundant at noradrenaline (fluorescent) cell-rich regions than at adrenaline (non-fluorescent) cell-rich regions. These results show that neurocalcin-immunoreactive nerves mainly innervate noradrenaline-containing chromaffin cells in the mouse adrenal medulla and that neurocalcin may regulate synaptic function in the nerve terminals. Received: 21 October 1996 / Accepted: 12 February 1997     

Substance P-containing axon terminals in the mucosa of the human urinary bladder: pre-embedding immunohistochemistry using cryostat sections for electron microscopy

The ultrastructure of substance P (SP)-containing axon terminals in the mucosa of the human urinary bladder was studied. Numerous SP-immunoreactive varicose nerve fibers were seen in the lamina propria, and most of them ran freely in the connective tissue. Many SP-immunoreactive nerve fibers were observed beneath the epithelium, and perivascular SP-immunoreactive nerves were also found in the submucosal layer. We observed a total of 305 SP-immunoreactive (IR) axon terminals, of which most (89.6%) were free nerve endings at the ultrastructural level; the rest of the SR-IR axon terminale were seen in the vicinity of the epithelium and blood vessels in the lamina propria. Varicose regions of SP-IR axon terminals contained large granular and small agranular synaptic vesicles, and most of them partially lacked a Schwann cell sheath. In some SP-IR varicosities, synaptic vesicles were concentrated in the region without any Schwann cell sheath. Long storage (for more than 1 month) of fixed-tissue pieces in sucrose before freezing has improved the ultrastructure of cryostat sections in pre-embedding immunohistochemistry. Trypsin digestion for the purpose of exposing antigenic sites was also employed before applying the first antiserum.     

Vesicular glutamate transporter 2-immunoreactive afferent nerve terminals in the carotid body of the rat

The carotid body is a peripheral chemoreceptor that detects decreases in arterial pO₂ and subsequently activates the carotid sinus nerve. The hypoxia-evoked activity of the carotid sinus nerve has been suggested to be modulated by glutamate. In the present study, we investigate the immunohistochemical localization of vesicular glutamate transporters in the carotid body of the rat. Vesicular glutamate transporter 2 (VGLUT2) labeling was closely associated with glomus cells immunoreactive to tyrosine hydroxylase but was not in the cytoplasm of these cells. The VGLUT2 immunoreactivity was observed within nerve endings that were immunoreactive to P2X3 and densely localized inside P2X3-immunoreactive axon terminals. These results suggest that VGLUT2 is localized in the afferent nerve terminals of the carotid body. Glutamate may be released from afferent nerve terminals to modulate the chemosensory activity of the carotid body.     

Light, fluorescence and electron microscopic studies of rabbit subclavian glomera.

The subclavian glomera (aortic bodies) of young New Zealand white rabbits were studied with the light, fluorescence, and electron microscopes. Two cell types were identified: type I, granule-containing (chief) cells, and type II, agranular (sustentacular) cells. The type I cells possessed large nuclei, the normal complement of cytoplasmic organelles and numerous electron-opaque cytoplasmic granules. The type II cells were agranular with attenuated cytoplasmic processes which partially or completely ensheathed the type I cells. The glomera were well vascularized. Capillary endothelial cells contained numerous pinocytotic vesicles, but few fenestrae. Two profiles of nerve terminals were observed. One, apposing the type I cells, contained numerous electron-lucent vesicles, several dense-cored vesicles, mitochondria and possessed membrane specializations resembling those usually observed in synaptic zones. The other profile contained abundant mitochondria and a few electron-lucent and dense-cored vesicles. Structural specializations were not observed on the apposed membranes of these terminals or adjacent to type II cells. Fluorescence histochemistry revealed an intense yellow-green fluorescence in the glomera, which indicated the presence of biogenic amines, possibly primary catecholamines or an indolamine. The electron-opaque granules observed in the type I cells were believed to be the storage sites for these amines. The subclavian glomera were found to be morphologically similar to the carotid body which is a known chemoreceptor.     

Demonstration of substance P in aortic nerve afferent fibers by combined use of fluorescent retrograde neuronal labeling and immunocytochemistry

Combined use of the intraaxonal retrograde transport of the fluorescent marker ‘true blue’ with substance P (SP) immunocytochemistry has been used to trace the nodose ganglion projections of SP-containing neurons of the aortic depressor nerve. It has been found that (1) SP immunoreactive (SP-I) cell bodies are clearly demonstrable in clusters in the rostral part of the nodose ganglion without the aid of colchicine pretreatment; (2) ‘true blue’ is retrogradely transported to the nodose ganglion following its application to the central cut end of the aortic nerve; (3) ‘true blue’ fluorescence and SP fluorescent immunoreactivity can be visualized in the same tissue section and certain cell bodies in the nodose ganglia contain both SP-I and retrogradely transported ‘true blue’. These results indicate that the aortic nerve which projects from the aortic arch baro- and/or chemoreceptors to brainstem vasomotor centers contains SP-I afferent fibers which emanate from the nodose ganglion.     

Localization of substance P,CGRP, VIP,neuropeptide Y,and somatostatin immunoreactive nerve fibers in the carotid labyrinths of some amphibian species

Summary Immunohistochemical localization of substance P (SP), CGRP, VIP, neuropeptide Y (NPY), and somatostatin (SOM) in the carotid labyrinth were compared in some species of amphibians using the peroxidase-antiperoxidase method. Immunoreactivity of SP, CGRP, VIP, and NPY was found in the nerve fibers distributed in the intervascular stroma of the carotid labyrinth. SP, CGRP, and VIP immunoreactive varicose fibers were densely distributed in the peripheral portion of the carotid labyrinth. Some SP-immunoreactive fibers were distributed similarly to CGRP-immunoreactive fibers. The density of NPY and SOM immunoreactive varicose fibers was low. No immunoreactivity of enkephalins was observed in the labyrinth. The intensities of these peptides were varied from species to species. No glomus cells showed immunoreactivity for any of the 7 peptides studied. These results suggest that the vascular regulatory function, which is one of the possible functions of the carotid labyrinth, is controlled by the peptidergic mechanisms in addition to regulation through intimate apposition of glomus and smooth muscle cells (g-s connection).     

Immunohistochemical localization of tryptophan hydroxylase and serotonin transporter in the carotid body of the rat

It has been proposed that serotonin (5-HT) facilitates the chemosensory activity of the carotid body (CB). In the present study, we investigated mRNA expression and immunohistochemical localization of the 5-HT synthetic enzyme isoforms, tryptophan hydroxylase 1 (TPH1) and TPH2, and the 5-HT plasma membrane transport protein, 5-HT transporter (SERT), in the CB of the rat. RT-PCR analysis detected the expression of mRNA for TPH1 and SERT in extracts of the CB. Using immunohistochemistry, 5-HT immunoreactivity was observed in a few glomus cells. TPH1 and SERT immunoreactivities were observed in almost all glomus cells. SERT immunoreactivity was seen on nerve fibers with TPH1 immunoreactivity. SERT immunoreactivity was also observed in varicose nerve fibers immunoreactive for dopamine beta-hydroxylase, but not in nerve fibers immunoreactive for vesicular acetylcholine transporters or nerve terminals immunoreactive for P2X₃ purinoreceptors. These results suggest that 5-HT is synthesized and released from glomus cells and sympathetic nerve fibers in the CB of the rat, and that the chemosensory activity of the CB is regulated by 5-HT from glomus cells and sympathetic nerve fibers.     

Localization of substance P, CGRP, VIP, neuropeptide Y, and somatostatin immunoreactive nerve fibers in the carotid labyrinths of some amphibian species.

Immunohistochemical localization of substance P (SP), CGRP, VIP, neuropeptide Y (NPY), and somatostatin (SOM) in the carotid labyrinth were compared in some species of amphibians using the peroxidase-antiperoxidase method. Immunoreactivity of SP, CGRP, VIP, and NPY was found in the nerve fibers distributed in the intervascular stroma of the carotid labyrinth. SP, CGRP, and VIP immunoreactive varicose fibers were densely distributed in the peripheral portion of the carotid labyrinth. Some SP-immunoreactive fibers were distributed similarly to CGRP-immunoreactive fibers. The density of NPY and SOM immunoreactive varicose fibers was low. No immunoreactivity of enkephalins was observed in the labyrinth. The intensities of these peptides were varied from species to species. No glomus cells showed immunoreactivity for any of the 7 peptides studied. These results suggest that the vascular regulatory function, which is one of the possible functions of the carotid labyrinth, is controlled by the peptidergic mechanisms in addition to regulation through intimate apposition of glomus and smooth muscle cells (g-s connection).     

Electron Microscopic Observations of the Carotid Body of the Cat

Carotid bodies were removed from cats, fixed in buffered 1 per cent osmic acid, embedded in deaerated, nitrogenated methacrylate, and cut into thin sections for electron microscopic study. The carotid body is seen to be composed of islands of chemoreceptor and sustentacular cells surrounded by wide irregular sinusoids. These cells are separated from the sinusoids by relatively broad interstitial spaces which are filled with collagen, fibroblasts, and many unmyelinated nerve fibers with their Schwann cell sheaths. The chemoreceptor cells are surrounded by the flattened, multiprocessed sustentacular cells which serve to convey the axons from an interstitial to a pericellular location. These sustentacular cells are assumed to be lemmoblastic in origin. Relatively few axons are seen to abut on the chemoreceptor cells. The cytoplasm of the chemoreceptor cell is characterized by numerous small mitochondria, units of granular endoplasmic reticulum, a small Golgi complex, and a variety of vesicles. There are many small vesicles diffusely scattered throughout the cytoplasm. In addition, there is a small number of dark-cored vesicles of the type which has been previously described in the adrenal medulla. These are usually associated with the Golgi complex. These findings are discussed in relation to the concepts of the origin of the chemoreceptor cell and the nature of the synapse.