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.     

Innervation of the human internal carotid arteries under normal conditions and in stenosis]

The neurohistological investigation of a portion of the internal carotid artery removed in operation on the occasion of occlusion revealed afferent, cholinergic and adrenergic nerve elements randomly located in the examined area. The histochemical and electronmicroscopic investigation of the superior cervical sympathetic ganglion removed from 42 patient operated on the occasion of occlusion of the carotid artery revealed a depletion in the ganglia of synaptic active zones, focal absence of catecholamines and neurohistological materials suggests that a substantial role in the process of stenosing of vascular walls is played by sophisticated effects of innervation connections upon the vessel sheaths.     

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.     

Sources of the innervation of the carotid reflexogenic zone in representative reptiles

The investigation is dedicated to study sources of the carotid reflexogenic zone innervation in 43 tortoises (Testudo horsfieldi and Emys orbicularis). In 7 tortoises fine preparation of the vessels and nerves of the cervical area after V. P. Vorob'ev has been performed. In 13 animals descending branch of the glossopharyngeal nerve has been resected. In 4--the caudal ganglion of this nerve and in 9 tortoises the caudal ganglion of the vagus nerve have been resected. In 10 tortoises adrenergic nervous plexuses are studied after Falck-Govyrin method, and cholinergic ones--after Karnovsky-Roots. As demonstrate anatomical investigations, to the carotid reflexogenic zone of the tortoises, situating in the area of the common carotid artery base, the branches of the glossopharyngeal and of the vagus nerve approach. The experiments with resection of these nervous conductors demonstrate that by the end of 3 days after the operation myelin nervous fibers of various thickness are at the stage of granular decay. Cholinergic and adrenergic nervous fibers and plexuses are revealed histochemically in the carotid zone.     

The neuronal and extra-neuronal localisations of biogenic amines in the cervical region of the domestic fowl (Gallus Gallus domesticus L.)

Summary The Falck-Hillarp technique has been used to demonstrate the neuronal and extra-neuronal localisations of biogenic amines in the cervical region of the domestic fowl. Adrenergic cell bodies were found in the superior cervical ganglion and in the ganglia of the cervical paravertebral chain. The axons of the latter ran into the corresponding spinal nerves and thus to the periphery. Very few adrenergic fibres were found in the interganglionic portions of the cervical paravertebral chain. The precarotid branch of the glossopharyngeal nerve, and the vagus nerve, below its junction with the former, contained numbers of adrenergic fibres. The retrocarotid nerve-trunk from the superior cervical ganglion was composed of adrenergic fibres. With the exception of the parathyroid gland, the adrenergic nerves seen in the branchial derivatives (thymus, thyroid and ultimobranchials) appeared to be associated with blood vessels. Under normal conditions the cells of the ultimobranchial body were nonfluorescent, but after injection of 6-hydroxydopamine the cells were brightly fluorescent. The carotid body was devoid of adrenergic nerves other than those with blood vessels, but the cells of the carotid body were brightly fluorescent. Various fluorescent cell types were found throughout the cervical region, particularly in association with the vasculature. I should like to thank Prof. G. Burnstock (Department of Zoology, Melbourne University) in whose department this work was carried out and Dr. R. D. Hodges (Wye College, London University) for his indispensable advice on the disposition of the avian ultimobranchial body. The author held a Postdoctoral Research Fellowship of the National Heart Foundation of Australia during part of this study.     

A comparative study of the distribution of carotid body type-I cells and periadventitial type-I cells in the carotid bifurcation regions of the rabbit,rat, guinea-pig and mouse

Summary The bilateral distribution of carotid body type-I cells was investigated in five rabbits, rats, guinea-pigs and mice by serially sectioning the carotid bifurcation regions. Carotid body type-I cells occurred bilaterally in close proximity to the wall of the internal carotid artery in the rabbit, rat and mouse and to the wall of the ascending pharyngeal artery in the guinea-pig. The rat carotid body was sometimes recessed into the lateral aspect of the superior cervical ganglion and was the most easily defined organ in the four animals studied. Caudally, and separate from the principal mass of carotid body type I cells, isolated groups of periadventitial type-I cells were observed in the connective tissues around the internal carotid artery and adjacent to the carotid bifurcation and common carotid artery in the rabbits only. An overall picture of the carotid body in the four animals was constructed. In all specimens rostral-caudal dimensions were recorded and compared bilaterally.The authors are indebted to Mr. Stephen Jones and Miss Alison Field of the Department of Histopathology, St Bartholomew's Hospital, for expert assistance in the preparation of the material; Miss J. McClelland and Miss C. Slatter for illustrations, and Mr. A. J. Aldrich and Mr. P.S. Hazell for photography. This work was supported by a grant from the Wellcome Trust to one of us (M. de B. D.)     

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.     

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

Summary 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.     

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.     

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.     

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.     

Cholinergic nature of the primary afferent vagus synapsed in cross anastomosed superior cervical ganglia.

The superior cervical ganglion was reinnervated by vagal afferent fibers following heterologous cross anastomosis between the superior cervical preganglionic trunk and the vagal trunk at the level of the supranodose ganglion in cats. The contractions of the nictitating membrane and the postganglionic action potentials from the external carotid sinus nerve in response to electrical stimulation of the vagal artificial preganglionic trunk in these operated cats were inhibited by treatment with tetraethylammonium and atropine. The choline acetyltransferase activities were measured by the radiometric method. The activities in cross anastomosed superior cervical ganglion were lower than those of normal superior cervical ganglion, but higher than those of chronically decentralized superior cervical ganglion. The activities in cross anastomosed nodose ganglion were lower than those of normal nodose ganglion, but higher than those of chronically decentralized superior cervical ganglion. These results further support the view that the primary afferent vagus artificially synapsed in the superior cervical ganglion is cholinergic.     

Regulation of Tyrosine Hydroxylase Gene Expression in the Rat Carotid Body by Hypoxia

The activity (Vmax) of tyrosine hydroxylase (TH; EC 1.14.16.2), the rate limiting enzyme in the synthesis of catecholamines, is increased in carotid body, superior cervical ganglion, and the adrenal medulla during hypoxia (i.e., reduced PaO2). The present study was undertaken to determine if the increase in TH activity in these tissues during hypoxia is regulated at the level of TH mRNA. Adult rats were exposed to hypoxia (10% O2) or room air for periods lasting from 1 to 48 h. The carotid bodies, superior cervical ganglia, and adrenals were removed and processed for in situ hybridization using 35S-labeled oligonucleotide probes. The concentration of TH mRNA was increased by hypoxia at all time points in carotid body type I cells, but not in cells of either superior cervical ganglion or adrenal medulla. The increase in TH mRNA in carotid body during hypoxia did not require innervation of the carotid body or intact adrenal glands. In addition, hypercapnia, another physiological stimulus of carotid body activity, failed to induce an increase in TH mRNA in type I cells. Our findings suggest that hypoxia stimulates TH gene expression in the carotid body by a mechanism that is intrinsic to type I cells.     

Conducting pathways of the superior cervical sympathetic ganglion of the cat

Individual nerves of the superior cervical sympathetic ganglion were stimulated in acute experiments on cats, and action potentials (AP) were recorded from other nerves of the ganglion in order to clarify whether or not there is transmission of excitation through the ganglion from one nerve to another and to establish whether this transmission is continuous or synaptic. The method of intracellular recording from neurons of the ganglion was also used. It is established that stimulation of the cervical sympathetic nerve evokes AP in all of the peripheral nerves of the ganglion, a circumstance that is the result of synaptic transmission of excitation. There is no transmission of excitation in the reverse direction or between any of the 12 peripheral nerves of the ganglion (including the four branches of the internal carotid nerve). Orthodromic excitation is recorded intracellularly from neurons of the ganglion during stimulation of the cervical sympathetic nerve, and antidromic excitation is recorded during stimulation of a peripheral nerve (the internal carotid nerve). It follows that the pathways through the ganglion which conduct excitation from the cervical sympathetic nerve into all of the remaining nerves of the ganglion are synaptic. Analysis of EPSP latent periods indicated that preganglionic fibers that differ sharply with respect to threshold and conduction rate (groups S₂ and S₄) converge on one and the same neurons of the ganglion.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 2, No. 2, pp. 216–224, March–April, 1970.     

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.     

Dopamine-β-Hydroxylase Activity of the Cat Carotid Body Under Different Arterial O2 and CO2 Conditions

To determine the importance of dopamine and noradrenaline as neurotransmitters during chemoreception in the cat carotid body we investigated the contents of both compounds as well as the activity of dopamine-beta-hydroxylase (DBH) under different arterial PO2 and PCO2 conditions. The superior cervical ganglion was used as a control organ. In the carotid body and the ganglion an inverse relationship exists between the catecholamine content and the DBH activity. The carotid body has a high catecholamine content with a low DBH activity whereas the superior cervical ganglion has a low catecholamine content and high DBH activity. Hypercapnia did not produce any significant change in the catecholamine content or in the DBH content of the carotid body. However, in comparison with hyperoxia, hypoxia produced a significant change (p less than 0.05) in the noradrenaline content without changing the DBH activity. The dopamine content under these conditions did not change significantly. The results may indicate that the high catecholamine content of the carotid body is the result of a high retention and/or low rate of degradation rather than of a high rate of synthesis.     

Effect of preganglionic sympathectomy on the cholinesterase activity in the superior cervical ganglia of rats and hamsters

Summary By employing biochemical assay and histochemical enzyme techniques the effect of preganglionic sympathectomy on the cholinesterase (ChE) activity in the superior cervical ganglia of rats and hamsters was investigated. Biochemical assays indicate that the ChE activity in the superior cervical ganglia of adult rats and hamsters is 57.19 and 28.63 respectively (expressed in u moles acetylcholine hydrolyzed per min per g of tissue); two weeks after preganglionic denervation, about 50% and 60% of ChE activity are lost respectively. Histochemical enzyme examination reveals that in the rat superior cervical ganglion, the majority of the neurons are adrenergic with weak to moderate acetylcholinesterase (AChE) reaction and the minority of the neurons are cholinergic with strong AChE activity, while only one type of adrenergic neurons exhibits a weak AChE activity in the hamster superior cervical ganglion. The AChE activity is localized in the perinuclear area, in the cisternae of the rough surfaced endoplasmic reticulum, in the Golgi complex and on the plasma membrane of the hamster's neurons; it is mainly localized in the cisternae of the rough surfaced endoplasmic reticulum of the rat's neurons. AChE reaction product is also detected on the axolemmal membranes of the preganglionic nerve fibers in the sympathetic ganglia of rats and hamsters.After preganglionic sympathectomy, the AChE activity in the adrenergic neurons and in the preganglionic unmyelinated nerve fibers is markedly reduced, whereas the cholinergic neurons and preganglionic myelinated nerve fibers remain unchanged. On the basis of these results two conclusions have been reached: (1) The fact that strong AChE activity localized in the cholinergic neurons and preganglionic myelinated fibers is not influenced by denervation, suggests that these structures are able to produce AChE. (2) The reduction of AChE activity in the rat and hamster superior cervical ganglia two weeks after preganglionic denervation, observed by histochemical examination, can be correlated with a concomitant measurable reduction determined by biochemical assays.Supported in part by a grant from the National Science Council, Republic of China. The author wishes to express his gratitude to the Department of Pharmacology, College of Medicine, National Taiwan University, for the use of its equipment for biochemical assays     

Perivascular nerves in the feline carotid rete

Summary Numerous nerve fibres containing acetylcholinesterase and noradrenaline, as well as avian pancreatic polypeptide-, vasoactive intestinal peptide-, or substance P-like immunoreactivity are observed around arteries in the external carotid rete of the cat. The nerves are located in the adventitial layer close to the media. It is possible that adrenergic, cholinergic and peptidergic nerve fibres may have a strong neurogenic influence on the rete blood vessels.     

Development of adrenergic neurons in uiuo: inhibition by ganglionic blockade

T he N ormal biochemical maturation of postsynaptic adrenergic neurons in mouse and rat superior cervical ganglion depends upon an intact preganglionic innervation (B lack , H endry and I versen , 1971a, 1972; T hoenen , S aner and K eitler , 1972). In recent studies tyrosine hydroxylase, the rate-limiting enzyme in norepinephrine biosynthesis (L evitt , S pector , S joerdsma and U denfriend , 1965), with localization to adrenergic neurons in the ganglion (B lack , H endry and I versen , 1971b), was used to monitor maturation of these cells. The developmental increase in tyrosine hydroxylase activity occurred simultaneously with the appearance of ganglionic synapses and was prevented by transection of the preganglionic nerve trunk (B lack , H endry and I versen , 1971a). These observations suggest that presynaptic cholinergic nerve terminals regulate the biochemical development of postsynaptic neurons in the superior cervical ganglion. The mechanism(s) by which presynaptic cholinergic terminals regulate postsynaptic development has not been elucidated. Such trans-synaptic regulation may be dependent on normal impulse transmission and/or may involve other unidentified, trophic factors. The results presented in the present communication suggest that normal development of ganglionic tyrosine hydroxylase activity is dependent on depolarization of postsynaptic adrenergic neurons.     

Fine structure of dense-cored vesicles in glomus cells of rat carotid body after fixation with permanganate or glutaraldehyde

Summary Glomus (Type I) cells of the carotid body of adult rats were studied electron microscopically after fixation with potassium permanganate or with glutaraldehyde and osmium tetroxide. Two permanganate fixation methods (using Krebs-Ringer-glucose, pH 7.0, or acetate buffer, pH 5.0) were compared. Numerous dense-cored vesicles were observed only in about one tenth of the glomus cells when neutral permanganate was used for fixation, although all glomus cells showed such vesicles after fixation with glutaraldehyde and osmium tetroxide. Numerous vesicles with a dense core were observed in about one third of the cells after fixation with acid potassium permanganate. With this fixation, small dense-cored vesicles similar to those in adrenergic nerve terminals were occasionally seen in the cytoplasm of glomus cells. It is tentatively concluded that the amine-storing vesicles of the carotid body are different from those in the small intensely fluorescent (SIF) cells and those in adrenergic nerve terminals.