Peptide-containing neurons projecting to the vocal cords of the rat: retrograde tracing and immunocytochemistry

The distribution and origin of neuropeptide Y-, vasoactive intestinal peptide- and calcitonin gene-related peptide-containing nerve fibers and adrenergic (dopamine-beta-hydroxylase-containing) fibers in the rat larynx were studied by retrograde tracing and selective denervations in combination with immunocytochemistry. An injection of the retrograde tracer True Blue to the right vocal cord resulted in the appearance of labelled nerve cell bodies in the ipsi- and contralateral superior cervical and stellate ganglia, the thyroid ganglia, the jugular-nodose ganglionic complexes, in the ipsilateral trigeminal and dorsal root ganglia at levels C2 and C3 and in local tracheal ganglia. Judging from the number of labelled nerve cell bodies, the jugular-nodose ganglionic complexes, dorsal root ganglia and superior cervical ganglia provide the greater part of the vocal cord innervation. Most of the True Blue-labelled nerve cell bodies in the superior cervical and stellate ganglia contained neuropeptide Y. In the thyroid ganglia the majority of labelled nerve cell bodies contained vasoactive intestinal peptide. In the jugular-nodose ganglionic complex and the dorsal root ganglia the majority of the labelled nerve cell bodies stored calcitonin gene-related peptide. Retrograde tracing and denervation studies revealed that all noradrenaline- and the majority of neuropeptide Y-containing nerve fibers emanate from the superior cervical and stellate ganglia. A minor population of neuropeptide Y-containing nerve fibers originate in local tracheal ganglia. The vasoactive intestinal peptide-containing nerve fibers originate in the thyroid ganglion and local tracheal ganglia, whereas calcitonin gene-related peptide-containing nerve fibres emanate from the dorsal root ganglia (C2-C3), the trigeminal ganglia and the jugular-nodose ganglia.     

Neuronal pathways to the rat middle meningeal artery revealed by retrograde tracing and immunocytochemistry

The origin of nerve fibers to the middle meningeal artery of rat was studied by retrograde tracing in combination with immunocytochemistry. Application of the retrograde tracer, True blue (TB), to the middle meningeal artery labeled nerve cell bodies in the ipsilateral superior cervical ganglion, the otic ganglion, the sphenopalatine ganglion, the jugular-nodose ganglionic complex, the trigeminal ganglion and the cervical dorsal root ganglion at level C2. A few nerve cell bodies were labeled in the contralaterally placed ganglia. Judging from the number of labeled nerve cell bodies, the ipsilateral superior cervical ganglion, the otic ganglion, the sphenopalatine and the trigeminal ganglia contribute most to its innervation. A moderate supply of labeled nerve cell bodies was seen in the cervical dorsal root ganglion at level C2 whereas there were only few in the jugular-nodose ganglionic complex. The TB-labeled nerve cell bodies were further examined for the presence of neuropeptides. For that purpose antibodies against neuropeptide Y (NPY), vasoactive intestinal polypeptide (VIP), calcitonin gene-related peptide (CGRP) and substance P (SP) were used. Most of the TB-labeled nerve cell bodies in the superior cervical ganglion exhibited NPY-immunoreactivity. In the sphenopalatine and otic ganglia the majority of the TB-labelled neurons were VIP-immunoreactive whereas in the trigeminal and dorsal root ganglion at level C2 the majority of labeled nerve cell bodies displayed CGRP-immunofluorescence and a minority of the labeled nerve cell bodies were substance P-immunoreactive. Together the findings indicate that several ganglia project to the middle meningeal artery of the rat and that many neuropeptides are involved in vasomotor control and in mediation of afferent information.     

Retrograde tracing of nerve fibers to the rat middle cerebral artery with true blue: colocalization with different peptides

The origin of nerve fibers to the rat middle cerebral artery was studied by retrograde tracing with the fluorescent tracer True Blue (TB) in combination with immunocytochemistry to known perivascular peptides. Application of TB to the middle cerebral artery labeled nerve cell bodies in the ipsilateral superior cervical ganglion, the otic ganglion, the sphenopalatine ganglion, the trigeminal ganglion, and the cervical dorsal root ganglion at level C2. A few labeled nerve cell bodies were seen in contralateral ganglia. Judging from the number and intensity of the labeling, the superior cervical ganglion and the trigeminal ganglion and dorsal root ganglion at level C2 contributed most to the innervation. A moderate number of nerve cell bodies were labeled in the sphenopalatine and otic ganglia. The TB-labeled nerve cell bodies were further examined for the presence of neuropeptides. For that purpose antibodies raised against neuropeptide Y (NPY), vasoactive intestinal polypeptide (VIP), substance P (SP) and calcitonin gene-related peptide (CGRP) were used. A considerable portion of the TB-labeled nerve cell bodies in the superior cervical ganglion contained NPY. About half of the labeled nerve cell bodies in the sphenopalatine and otic ganglia contained VIP. In the trigeminal ganglion and in the dorsal root ganglion at level C2, one-third of the TB-labeled nerve cell bodies were CGRP-immunoreactive, while only few nerve cell bodies contained SP. The study provides direct evidence for the origin of cerebrovascular peptidergic nerve fibers and demonstrates that not only ipsilateral but also contralateral ganglia contribute to the innervation of the cerebral circulation.     

Peptide-containing Neurons Projecting to the Tongue of the Rat: Retrograde Tracing and Immunocytochemistry

The origin and neuropeptide content of nerve fibres in the rat circumvallate papilla was studied by retrograde tracing in combination with immunocytochemistry. An injection of the retrograde tracer True Blue into the circumvallate papilla resulted in the appearance of labelled nerve cell bodies in the superior cervical, the stellate, the thyroid, the nodose, the jugular, the petrosal, the otic, the trigeminal and the dorsal root ganglia at level C2. Most of the True Blue-labelled nerve cells in the superior cervical ganglia contained neuropeptide Y. The majority of labelled cell bodies in the thyroid ganglia contained vasoactive intestinal peptide. In the jugular and trigeminal ganglia, the majority of the labelled nerve cell bodies stored calcitonin gene-related peptide. A small number of neurons in the medial reticular formation of the central nervous system was labelled. Tracer injections deep into the tongue tissue beneath the circumvallate papilla gave rise to True Blue-labelled neurons in the hypoglossal nucleus.     

Dual intrinsic and extrinsic origins of CGRP- and NPY-immunoreactive nerves of rat gut and pancreas

The origins of nerves containing calcitonin gene-related peptide (CGRP), neuropeptide Y (NPY), and substance P were investigated in the rat stomach, pancreas, and colon, using immunocytochemistry combined with retrograde tracing and surgical and chemical denervation procedures. Compared with nerves containing vasoactive intestinal polypeptide (VIP) and galanin, which have primarily local origins in mammalian gut, CGRP-, NPY-, and substance P-immunoreactive nerves revealed dual extrinsic and intrinsic origins. Immunocytochemistry combined with retrograde tracing showed that the extrinsic CGRP- and substance P-immunoreactive nerves in the stomach and pancreas originate from bilateral dorsal root ganglia mainly at levels T8-T11, while those of the colon are derived from bilateral ganglia at S1 and, to a lesser extent, L1 and L6. Chemical denervations showed that neurons in these ganglia form a sensory input to the gut, and that those containing CGRP form the largest proportion. The results of combined retrograde tracing and immunocytochemistry indicated that extrinsic NPY-immunoreactive nerves originate from postganglionic sympathetic neurons lying in the coeliac and inferior mesenteric ganglia. These nerves were located mainly around blood vessels in gut and pancreas, showed sensitivity to 6-hydroxydopamine, and thus are likely to be noradrenergic. The present study provides a detailed mapping of the origins of some of the major peptide-containing nerves of the rat gastroenteropancreatic tract, thus providing further information on the anatomy of the enteric innervation.     

Neuropeptides in the human superior cervical ganglion

Superior cervical ganglia from 7 human cadavers (3–7 h post mortem) were immunostained for tyrosine hydroxylase (TH), dopamine-β-hydroxylase (DBH) and 14 different neuropeptides. The results show that ganglionic cells contain TH, DBH, neuropeptide Y (NPY), somatostatin, vasoactive intestinal polypeptide (VIP) and calcitonin gene-related peptide (CGRP). These substances were present predominantly within large ganglionic cells. Inside the ganglion, the number and topographical distribution of various types of immunoreactive cells differed from one another. NPY and CGRP immunoreactivities were found in some TH-positive cells, but that co-localization never exceeded the 30% of the TH cells. Leu-enkephalin showed a weak immunoreactivity, which was restricted to fibers or varicosities. Neuropeptides like substance P, dynorphin A and B, cholecystokinin, galanin, corticotropin-releasing factor, thyreotropin-releasing hormone, angiotensin II and neurotensin showed no immunoreactivity in the human superior cervical ganglion.     

Local differences in vagal afferent innervation of the rat esophagus are reflected by neurochemical differences at the level of the sensory ganglia and by different brainstem projections

The objective of the present study was to characterize further the vagal afferent fibers in the rat esophagus, particularly those in its uppermost part, their cell bodies in vagal sensory ganglia, and their central projections. We applied immunohistochemistry for calretinin, calbindin, and calcitonin gene-related peptide (CGRP); retrograde tracing with FluoroGold; and transganglionic tracing with wheat germ agglutinin-horseradish peroxidase in combination with neurectomies. Vagal terminal structures in the muscularis propria of the whole esophagus consisted of calretinin-immunoreactive intraganglionic laminar endings that were linked to cervical vagal and recurrent laryngeal nerve pathways. The mucosa of the uppermost esophagus was innervated by a very dense net of longitudinally arranged, calretinin-positive fibers that were depleted by section of the superior laryngeal nerve. Distal to this area, the mucosa was virtually devoid of calretinin-immunoreactive vagal afferents. Calretinin-positive mucosal fibers in the upper cervical esophagus were classified into four types. One type, the finger-like endings, was sometimes immunoreactive also for CGRP. About one-third of cell bodies in vagal sensory ganglia retrogradely labeled from the upper cervical esophagus expressed CGRP, whereas two-thirds coexpressed calretinin and calbindin but not CGRP. In addition to the central subnucleus of the nucleus of the solitary tract, vagal afferents from the upper cervical esophagus also projected heavily to the interstitial subnucleus. This additional projection was attributed to mucosal afferents traveling through the superior laryngeal nerve. The present study provides a possible morphological basis for bronchopulmonary and aversive reflexes elicited upon stimulation of the esophagus.     

Vasoactive peptide release in the extracerebral circulation of humans during migraine headache.

The innervation of the cranial vessels by the trigeminal nerve, the trigeminovascular system, has recently been the subject of study in view of its possible role in the mediation of some aspects of migraine. Since stimulation of the trigeminal ganglion in humans leads to facial pain and flushing and associated release of powerful neuropeptide vasodilator substances, their local release into the extracerebral circulation of humans was determined in patients who had either common or classic migraine. Venous blood was sampled from both the external jugular and cubital fossa ipsilateral to the side of headache. Plasma levels of neuropeptide Y, vasoactive intestinal polypeptide, substance P, and calcitonin gene-related peptide were determined using sensitive radioimmunoassays for each peptide, and values for the cubital fossa and external jugular and a control population were compared. A substantial elevation of the calcitonin gene-related peptide level in the external jugular but not the cubital fossa blood was seen in both classic and common migraine. The increase seen in classic migraine was greater than that seen with common migraine. The other peptides measured were unaltered. This finding may have importance in the pathophysiology of migraine.     

Perivascular noradrenergic and peptide-containing nerves show different patterns of changes during development and ageing in the guinea-pig

The development of noradrenergic and peptide-containing perivascular nerves in common carotid, mesenteric, renal and femoral arteries of the guinea-pig was studied using the glyoxylic acid fluorescence and indirect immunofluorescence techniques on whole-mount stretch preparations at 6 stages between 6 weeks in utero and two years after birth. The noradrenergic plexus was more dense than the peptide-containing nerve plexuses in all the blood vessels, and, in general, calcitonin gene-related peptide-containing nerves were more numerous than substance P-containing nerves which in turn were more numerous than vasoactive intestinal polypeptide-containing nerves. In mesenteric and carotid arteries, noradrenergic nerve density reached a peak at about 4 weeks after birth that was maintained to old age, whereas the peptide-containing vasoactive intestinal polypeptide (VIP) and calcitonin gene-related peptide (CGRP) nerve plexuses reached a peak at birth and declined thereafter to about half maximum density in old age. In contrast, in the renal and femoral arteries, peptide-containing nerves reached a maximum density at 4 weeks after birth, while noradrenergic nerve density reached a peak around birth; both noradrenergic and peptide-containing nerve plexuses declined in density in old age. Of the 4 vessels studied, the mesenteric artery showed the greatest density of innervation for both noradrenergic and peptide-containing nerves at all stages of development, while the femoral artery was the least innervated. The possibility that some perivascular peptide-containing nerves play a trophic role during development is discussed.     

Extrinsic pathways of the adrenergic innervation of the guinea-pig trachealis muscle

Origins and extrinsic pathways of the adrenergic innervation of the guinea-pig trachealis muscle were studied using fluorescence histochemical techniques. Bilateral superior cervical ganglionectomy caused a marked reduction in the adrenergic innervation of the extra-thoracic region, which suggests that these ganglia are a major source of adrenergic innervation to this muscle. Combined anterior and posterior transection of the recurrent laryngeal nerves also caused a marked reduction in the density of adrenergic fibres in the extra-thoracic trachealis muscle. Crushing of these nerves revealed adrenergic fibres running both anteriorly and posteriorly. The majority of these adrenergic nerves were lost after superior cervical ganglionectomy and thus the fibres running in both directions originate in the superior cervical ganglion. Antero-posteriorly directed fibres entered the recurrent laryngeal nerve from the superior cervical ganglion via an anastomosis at the level of the cricoid cartilage, while those running postero-anteriorly entered the recurrent laryngeal nerve posteriorly from the vagus nerve and these adrenergic fibres were lost after cervical vagotomy.     

Galanin immunoreactivity in autonomic innervation of the cat eye

In an immunohistochemical study, we find that galanin is much more widely distributed in the peripheral innervation of the cat eye than in other animals so far examined. Previous studies of rat and pig eyes have revealed sparse galanin-positive nerves that presumbably originate in the trigeminal ganglion. In contrast, the cat has a rich supply of galanin-containing nerve fibers throughout the uvea. Galanin-positive varicose nerves concentrate densely in iris muscles and distribute more sparsely in the ciliary muscle. The ciliary processes have a plexus of galanin-positive nerve underlying the ciliary epithelium at their base and positive nerve fibers coursing within their stroma. The ciliary artery and its branch vessels in the uvea are invested with a dense plexus of galanin-positive nerves. All autonomic ganglia supplying the eye contain cells that express galanin. It is present in 97% of superior cervical ganglion cells, coexisting with both tyrosine hydroxylase and neuropeptide Y; in 80% of pterygopalatine ganglion cells, most of whiclh also contain vasoactive intestinal peptide; and in approximately 25% of ciliary ganglion celis. After unilateral superior cervical ganglionectoiny, galanin-positive nerves almost totally disappear from the iris muscles, demonstrating that they are predominantly of sympathetic origin. Galanin-positive nerves investing the ciliary artery and choroidal blood vessels are not detectably reduced by sympathectomy, indicating that perivascular parasympathetic nerves from the pterygopalatine ganglion also express galanin. Other galanin-containing nerves in the eye can originate from the trigeminal and ciliary ganglia. The prominence of galanin in the ocular autonomic innervation of the cat provides an opportunity to explore the physiological effects of this neuropeptide in the eye. © 1994 Wiley-Liss, Inc.     

Different reinnervation patterns in the celiac/mesenteric and superior cervical ganglia following guanethidine sympathectomy in adult rats

We sought to determine whether chronic guanethidine (Gu) treatment in adult rats produces depletion of sympathetic neurons and hyperinnervation by sensory neuropeptides in the celiac/superior mesenteric (C/SMG) ganglion. Rats received Gu 40 mg/kg per day i.p or saline for 5 weeks. Upon completion of treatment, the C/SMG and the superior cervical ganglion (SCG) were examined for neuropeptide Y (NPY), substance P (SP), calcitonin gene-related peptide (CGRP) and vasoactive intestinal polypeptide (VIP), both by immunocytochemistry (ICC) and radioimmunoassay (RIA). Gu produced marked depletion of NPY-containing neurons and NPY content in the C/SMG, similar to that in the SCG (−89 ± 2vs.−92 ± 4%, respectively). SP and CGRP immunoreactivities were significantly higher iontrol C/SMG as compared with SCG; after Gu treatment, there was no significant increase in either SP or CGRP in the C/SMG, however, both increased in the SCG. In contrast, VIP levels were similar in the SCG and C/SMG in controls and increased in the C/SMG but not in the SCG after Gu treatment. Thus, in adult rats, the C/SMG is as susceptible as the SCG to Gu treatment; the different pattern of hyperinnervation by SP, CGRP and VIP of the C/SMG as compared with the SCG may reflect the different sources for these neuropeptides in prevertebral as compared with paravertebral ganglia.     

Pituitary adenylate cyclase-activating polypeptide-immunoreactive nerve fibers in rat and human tooth pulps

The distribution of pituitary adenylate cyclase-activating polypeptide (PACAP) was examined in the tooth pulp. In rat and human tooth pulps, PACAP-immunoreactive (IR) nerve fibers were observed around blood vessels and in the subodontoblastic and odontoblastic layers. The predentine and dentine were devoid of such nerve fibers. The double immunofluorescence method indicated the co-expression of PACAP with calcitonin gene-related peptide (CGRP) or vasoactive intestinal polypeptide (VIP). Virtually all PACAP-IR nerve fibers co-expressed CGRP-immunoreactivity (IR) in the rat tooth pulp suggesting their sensory function. In addition, a retrograde tracing method indicated that PACAP-IR nerve fibers in the rat tooth pulp originated from the trigeminal ganglion. On the other hand, almost all PACAP-IR nerve fibers in the human tooth pulp co-expressed VIP-IR and, thus, thought to be autonomic in nature.     

The origin and development of the vagal and spinal innervation of the external muscle of the mouse esophagus

Retrograde and anterograde tracing and immunohistochemical techniques were used to examine the origin of the extrinsic innervation, and the development of the vagal innervation to the mouse esophagus. Cholinergic nerve terminals were localised using an antiserum to the vesicular acetylcholine transporter and cholinergic cell bodies were localised using an antiserum to choline acetyltransferase. Cholinergic nerve terminals, which also contained calcitonin gene-related peptide, were present at the motor end plates in the external (striated) muscle of the esophagus. Following injection of Fast Blue into subdiaphragmatic or cervical levels of the esophagus, the only retrogradely-labelled cholinergic nerve cell bodies that also contained calcitonin gene-related peptide were found in the nucleus ambiguus. Neurons in the dorsal motor nucleus of the vagus, the nodose ganglia and dorsal root ganglia gave rise to a number of different types of nerve terminals within the myenteric plexus. Retrogradely-labelled neurons in the dorsal motor nucleus of vagus contained cholinergic markers only, nitric oxide synthase only or cholinergic markers plus nitric oxide synthase, retrogradely-labelled neurons in the dorsal root ganglia contained calcitonin gene-related peptide only, and a small number of retrogradely-labelled neurons in the nodose ganglia contained tyrosine hydroxylase. The development of the vagal innervation to the esophagus was examined following application of DiI to the vagus nerve of fixed mouse embryos. Anterogradely-labelled nerve fibres, which arose from both nodose ganglia and the medulla, were already present in the esophagus of embryonic day 12 (E12) mice. Some of the DiI-labelled vagal nerve fibres were present in among the smooth muscle cells of the external muscle layer prior to their transdifferentiation to striated muscle. We conclude that the neurons in the nucleus ambiguus that project to the esophagus differ from other extrinsic neurons in their chemistry as well as their targets within the esophagus. The development of the extrinsic innervation precedes the transdifferentiation of the external muscle to striated muscle, raising the possibility that, during development, smooth muscle of the esophagus is innervated transiently by vagal neurons.     

Accessory innervation of mandibular anterior teeth in cats: a horseradish peroxidase study

The substance P innervation of the rat parotid was investigated using retrograde tracing with the fluorescent dye True Blue, in combination with the immunohistochemical localization of substance P. Retrogradely-labelled cells were seen in the otic, trigeminal, superior cervical and nodose ganglia. A proportion of the True Blue-labelled cells in the otic and trigeminal ganglia also contained substance P-like immunoreactivity providing direct evidence for a dual origin of substance P in the rat parotid. In addition, these results imply that in the rat otic ganglion substance P and acetylcholine coexist.     

Retrograde tracing shows that CGRP-immunoreactive nerves of rat trachea and lung originate from vagal and dorsal root ganglia

The origins of sensory innervation of the lower respiratory tract are thought to be principally the nodose and jugular ganglia of the vagus nerve. It has been suggested and partially demonstrated that there is also a component arising from dorsal root ganglia, but the segmental levels involved are not known precisely. We have therefore investigated the origins of sensory nerves within the rat respiratory tract, particularly those containing calcitonin gene-related peptide (CGRP), using the technique of retrograde axonal tracing combined with immunohistochemistry. Injections of True blue were made into extra-thoracic trachea (n = 4 rats) and percutaneously into the right and left lung (n = 4 each). Retrogradely labelled neuronal perikarya were detected in vagal and dorsal root ganglia, and sympathetic chain ganglia. CGRP-immunoreactive cells were seen only in vagal and dorsal root ganglia. Tracheal innervation arose bilaterally in the vagal sensory ganglia but those on the right side represented the principal source; the majority of CGRP-containing neurons occurred in the jugular ganglion. A very small component of labelling occurred in spinal ganglia at levels C2-C6. The sensory innervation of the lungs was seen to arise predominantly from the ipsilateral dorsal root ganglia (45% of cells CGRP-immunoreactive) at levels T1-T6. In contrast to the trachea, the contribution of vagal sensory neurones to the lungs appeared to be less than that of the spinal ganglia. These results show that the sensory innervation of the rat lungs has a major origin in the dorsal root ganglia, in which almost half of the involved neurons contain CGRP, and confirm that most CGRP-immunoreactive nerves in the trachea arise in the right jugular ganglion.     

Substance P in the rat parotid gland: evidence for a dual origin from the otic and trigeminal ganglia

The substance P innervation of the rat parotid was investigated using retrograde tracing with the fluorescent dye True Blue, in combination with the immunohistochemical localization of substance P. Retrogradely-labelled cells were seen in the otic, trigeminal, superior cervical and nodose ganglia. A proportion of the True Blue-labelled cells in the otic and trigeminal ganglia also contained substance P-like immunoreactivity providing direct evidence for a dual origin of substance P in the rat parotid. In addition, these results imply that in the rat otic ganglion substance P and acetylcholine coexist.     

Satellite cells as blood-ganglion cell barrier in autonomic ganglia

In a preliminary study a difference in the uptake and transport of wheat germ agglutinin-horseradish peroxidase (WGA-HRP) between the trigeminal ganglion and the superior cervical ganglion was observed. After injection of WGA-HRP and HRP into the trigeminal ganglion, peroxidase was found in the space between the satellite cell processes and the ganglion cells. The ganglion cells showed pinocytosis and uptake of WGA-HRP and HRP. In the superior cervical ganglion WGA-HRP and HRP were found alongside the satellite cells but were absent in the space between satellite cells and ganglion cells. Intravenous injection revealed the presence of HRP in the space between sensory ganglion cells and their satellite cells of the trigeminal and nodose ganglion whereas HRP was absent in the space between autonomic ganglion cells and their satellite cells of the superior cervical, medial cervical and pterygopalatine ganglion although HRP lined the satellite cell membranes. By means of electron microscopy, satellite cell processes in the superior cervical ganglion were found to enwrap ganglion cells very tightly with a marginal space between both cell types. Satellite cells and their processes were mutually anchored by numerous tight junctions. In the trigeminal ganglion the extracellular space between ganglion cells and satellite cells was larger and satellite cells were found to be more loosely arranged around the ganglion cells. Satellite cell processes were only occasionally linked by tight junctions. It is concluded that satellite cells in autonomic ganglia comprise an effective barrier for WGA-HRP and HRP and probably large molecules in general. This barrier is absent in sensory ganglia.