Calcitonin Gene-related Peptide-containing Fibers Research Articles

Neurochemical plasticity of the carotid body in hypertension.

The carotid body (CB), a main peripheral arterial chemoreceptor, has lately been implicated in the pathophysiology of various cardiovascular disorders. Emerging experimental evidence supports a causal relationship between CB dysfunction and augmented sympathetic outflow which is the common hallmark of human sympathetic-related diseases, including essential hypertension. To gain insight into the neurotransmitter profile of chemosensory cells in the hypertensive CB, we examined the expression and cellular localization of some classical neurotransmitters, neuropeptides, and gaseous signaling molecules as well as neurotrophic factors and their receptors in the CB of spontaneously hypertensive rats, a common animal model of hypertension. Our immunohistochemical experiments revealed an elevated catecholamine and serotonin content in the hypertensive CB compared to normotensive controls. GABA immunostaining was seen in some peripherally located glomus cells in the CB of SHR and it was significantly lower than in control animals. The density of substance P and vasoactive intestinal peptide-immunoreactive fibers was diminished whereas that of neuropeptide Y-immunostained nerve fibers was increased and that of calcitonin gene-related peptide-containing fibers remained almost unchanged in the hypertensive CB. We have further demonstrated that in the hypertensive state the production of nitric oxide is impaired and that the components of the neurotrophin signaling system display an abnormal enhanced expression. Our results provide immunohistochemical evidence that the altered transmitter phenotype of CB chemoreceptor cells and the elevated production of neurotrophic factors modulate the chemosensory processing in hypertensive animals which contributes to autonomic dysfunction and elicits sympathetic hyperactivity, consequently leading to elevated blood pressure.

Increased responsiveness of rat colonic splanchnic afferents to 5‐HT after inflammation and recovery

5-Hydroxytryptamine (5-HT) activates colonic splanchnic afferents, a mechanism by which it has been implicated in generating symptoms in postinfectious and postinflammatory states in humans. Here we compared mechanisms of colonic afferent activation by 5-HT and mechanical stimuli in normal and inflamed rat colon, and after recovery from inflammation. Colonic inflammation was induced in rats by dextran sulphate sodium. Single-fibre recordings of colonic lumbar splanchnic afferents revealed that 58% of endings responded to 5-HT (10(-4) m) in controls, 88% in acute inflammation (P<0.05) and 75% after 21 days recovery (P < 0.05 versus control). Maximal responses to 5-HT were also larger, and the estimated EC50 was reduced from 3.2 x 10(-6) to 8 x 10(-7) m in acute inflammation and recovered to 2 x 10(-6) m after recovery. Responsiveness to mechanical stimulation was unaffected. 5-HT3 receptor antagonism with alosetron reduced responses to 5-HT in controls but not during inflammation. Responses to the mast cell degranulator 48/80 mimicked those to 5-HT in inflamed tissue but not in controls, and more 5-HT-containing mast cells were seen close to calcitonin gene-related peptide-containing fibres in inflamed serosa. We conclude that colonic serosal and mesenteric endings exhibit increased sensitivity to 5-HT in inflammation, with both an increase in proportion of responders and an increase in sensitivity, which is maintained after healing of inflammation. This is associated with alterations in the roles of 5-HT3 receptors and mast cells.

Sensory nerves affect the recruitment and differentiation of rat periovarian brown adipocytes during cold acclimation.

Rat periovarian adipose tissue contains a low number of uncoupling protein-expressing brown adipocytes scattered into lobules of white fat. Their increase following cold acclimation is matched by a major increase in noradrenergic and neuropeptide Y-, substance P- and calcitonin gene-related peptide-containing nerves. To ascertain whether periovarian fat is provided with sensory nerves, and whether any relationship exists between such nerves (in particular the calcitonin gene-related peptide-containing fibers found in cold-acclimated rats in close association with brown adipocytes) and brown fat recruitment, the effects of capsaicin desensitization on neuropeptide-containing nerves and brown adipocyte density were studied in the periovarian tissue of rats kept at 20 degrees C and on a group acclimated to 4 degrees C for 14 days. In both groups, systemic capsaicin administration considerably reduced the expression of substance P and calcitonin gene-related peptide in vascular-nerve bundles and parenchyma. In cold-acclimated rats, the increase in brown adipocyte density was significantly checked by capsaicin administration (21.11 versus 7.96 brown adipocytes/mm2, P<0.05). Finally, ultrastructural investigation showed the occurrence of brown adipocyte precursors filled with aggregates of glycogen and poorly differentiated multilocular adipocytes in capsaicin-treated cold-acclimated rats. These data suggest that periovarian adipose tissue is indeed provided with sensory neuropeptide-containing nerves and that they play a role in the recruitment and differentiation of brown adipocytes.

An ultrastructural study of calcitonin gene-related peptide-immunoreactive nerve fibers innervating the rat posterior longitudinal ligament. A morphologic basis for their possible efferent actions.

The present study investigated ultrastructural characteristics of calcitonin gene-related peptide-immunoreactive nerve fibers in the posterior longitudinal ligament of the rat lumbar spine. To provide a morphologic basis for assessment of the afferent and, in particular, efferent functions of calcitonin gene-related peptide immunoreactive nerves in the posterior longitudinal ligament and their eventual role in degenerative spondylarthropathies and low back pain. Previous studies using light-microscopic localization of sensory neuronal markers such as calcitonin gene-related peptide have reported the presence of sensory fibers in the supporting structures of the vertebral column. Meanwhile, accumulating research data have suggested efferent properties for calcitonin gene-related peptide, i.e., a trophic action that alters the intrinsic properties of target cells not through transient action of synaptic transmission, but through long-lasting signal transmission by the secreted neuropeptides. To verify such trophic, paracrine actions of the calcitonin gene-related peptide-containing fibers in the posterior longitudinal ligament, however, ultrastructural details of the terminals and their spatial relationship to their eventual target structures have to be elucidated. Rat posterior longitudinal ligaments were stained immunohistochemically for calcitonin gene-related peptide. Light-microscopic analysis of the semithin sections facilitated subsequent electron microscopy of specific sites of the posterior longitudinal ligament to determine ultrastructural details and nerve fiber-target relationships. The rat lumbar posterior longitudinal ligament was found to be innervated by two distinctive calcitonin gene-related peptide immunoreactive nerve networks. In immunoelectronmicroscopy, the fibers of the deep network had numerous free nerve endings, whereas those of the superficial network showed spatial associations with other non-calcitonin gene-related peptide immunoreactive components of the network. In both systems, naked axons not covered by the Schwann cells made close spatial contact with smooth muscle cells: of blood vessels and resident posterior longitudinal ligament fibroblasts. The ultrastructural characteristics of the innervation of the rat posterior longitudinal ligament would be compatible not only with a nociceptive function, but also with neuromodulatory, vasoregulatory, and trophic functions, as has already been established in some visceral organs.

An immunohistochemical study of sensory and autonomic innervation of the dog tongue with special reference to substance P- and calcitonin gene-related peptide-containing fibers in blood vessels and the intralingual ganglia.

The distribution, pathways and origins of peptide-containing nerve fibers in the anterior two thirds of the dog tongue were investigated using immunohistochemistry combined with retrograde axonal tracing and denervation experiments. Within the epithelium of the fungiform papillae, varicose nerve fibers immunoreactive to substance P (SP) and calcitonin gene-related peptide (CGRP) were present. These disappeared completely after severance of the lingual nerve (LN) alone. Dense CGRP-immunoreactive varicose fibers surrounded cell bodies in the intralingual ganglia (ILG), which consisted of neurons immunoreactive to vasoactive intestinal polypeptide (VIP), neuropeptide Y (NPY) and SP. These CGRP-immunoreactive fibers disappeared following severance of the chorda tympani (CT) alone. SP-, CGRP-, VIP-, NPY- and tyrosine hydroxylase (TH)-immunoreactive nerve fibers were distributed around the walls of blood vessels, especially arteriovenous anastomoses (AVAs). None of these immunoreactive fibers completely disappeared after severance of the LN or CT alone, but SP- and CGRP-immunoreactive fibers disappeared following severance of both the LN and CT. TH-immunoreactive fibers disappeared after ganglionectomy of the superior cervical ganglion (SCG) or severance of the hypoglossal nerve (HGN). VIP- and NPY-immunoreactive fibers invariably remained after various denervation experiments. In tracing experiments, CGRP-immunoreactive as well as SP and CGRP-immunoreactive cells in the trigeminal ganglion were labelled from the LN, and those in the geniculate ganglion and jugular ganglion were labelled from the CT. A large number of neurons in the SCG were labelled from the HGN, with some of these being SP and CGRP-immunoreactive. These results demonstrate that SP- and CGRP-immunoreactive fibers from the trigeminal ganglion are distributed to the lingual epithelium; vascular walls receive SP- and CGRP-immunoreactive sensory fibers from the LN as well as CT, some SP and CGRP-immunoreactive fibers from the SCG in addition to catecholaminergic sympathetic fibers, and VIP- and NPY-immunoreactive parasympathetic fibers from the ILG. The ILG is also considered to be innervated by CGRP-immunoreactive sensory fibers from the CT.

Ultrastructural Characterization of Calcitonin Gene-Related Peptide-Containing Fibers and Islet Cells in the Rat Pancreas

Morphology and structural organization of calcitonin gene-related peptide (CGRP) immunoreactive nerve fibers and islet cells in rat pancreas were analyzed with light and electron microscopic immunocytochemistry. Immunoreactive axons innervate exocrine and endocrine parenchyma, but are most abundant in connective tissue septae between pancreatic lobules and within the perivascular space of small arterioles. In the stromal compartment and perivascular space, immunoreactive product is confined to thin, unmyelinated axons, which represent a prominent component of large nerve bundles, composed of numerous, other, unlabeled axons and dendrites. Immunoreactive axons and terminals display multiple varicosities, filled with lucent spherical vesicles (40 nm average diameter), and are often in direct contact with unlabeled dendrites, presumed to arise from intrinsic pancreatic neurons. However, definitive synaptic contacts involving immunoreactive axon terminals were never observed, nor was CGRP immunoreactivity ever detected in neuronal cell bodies within intrapancreatic ganglia. Cellular immunoreactivity is relegated to perikarya at the peripheral margin of each islet of Langerhans, which emit one or more short, thick processes, which often terminate upon fenestrated capillaries. Immunoreaction product in these cells is concentrated in large secretory vesicles (approximately 230 nm diameter), which are dispersed throughout the somata and frequently appear to open into the perivascular space of capillaries. Immunoreactive axons, innervating the islets, are sparse and do not appear to have preferential association with immunoreactive cells. Present findings provide further evidence for a dual role of CGRP in pancreatic functions via a neuronal pathway and hormonal mechanisms.

Peptide-containing nerve fibers in the circumvallate papillae

The occurrence and distribution of an array of neuropeptides and dopamine-β-hydroxylase in the circumvallate papillae of monkey, pig, cow, ferret, cat, rat and mouse was studied by immunocytochemistry. The animals were chosen to represent species with different diets. Substance P/neurokinin A- and calcitonin gene-related peptide-containing fibers were numerous in the circumvallate papillae of all animals examined, with the highest frequency in monkey, pig, cow, rat and mouse; in ferret and cat moderate numbers were detected. Vasoactive intestinal peptide/peptide histidine isoleucine amide-containing fibers were numerous in the circumvallate papillae of pig, while they were moderate in number in monkey, ferret and mouse. Neuropeptide Y-containing fibers were few to moderate in number in the circumvallate papillae of all species. Galanin-containing fibers were numerous in the pig circumvallate papillae, while only a few fibers could be detected in monkey, cow, cat, rat and mouse. Somatostatin-containing fibers were seen only in the cat circumvallate papillae, gastrin-releasing peptide-containing fibers in the cow and cat, cholecystokinin/gastrin-containing fibers in the pig and cow. Dopamine-β-hydroxylase-containing fibers were detected in all animals studied. They were few to moderate in number in the circumvallate papillae. There was no obvious link between the peptidergic innervation pattern and the food habits.

Localization, origin and fine structure of calcitonin gene-related peptide-containing fibers in the vestibular end-organs of the rat

The localization, origin and fine structure of nerve terminals immunoreactive for calcitonin gene-related peptide (CGRP) were examined in the vestibular end-organs of rats using immunocytochemistry. Many CGRP-like immunoreactive (CGRP-IR) fibers were observed in the vestibular sensory epithelial layer. By electronmicroscopy, CGRP-IR terminals were found to make synaptic contacts with the chalyces of the vestibular nerves terminating on type I cells. The origin of these vestibular CGRP-IR fibers was examined by a combination of a retrograde fiber tracing technique (using Fast blue) and immunocytochemistry. Injections f the tracer into the vestibular cistern, resulted in fast blue-labeled neurons bilaterally in the area dorsolateral to the genu of the facial nerve; these labeled neurons also contained CGRP. These findings indicate that CGRP-IR fibers originate bilaterally from the area dorsolateral to the genu of the facial nerve and that CGRP plays a modulatory role in the transmission of vestibular information from type I cells.

Neuronal pathways to the rat thyroid revealed by retrograde tracing and immunocytochemistry

The distribution and origin of the nerve fibres innervating the rat thyroid were studied by immunocytochemistry, retrograde tracing and denervation experiments. Immunocytochemistry revealed nerve fibres containing noradrenalin, neuropeptide Y, vasoactive intestinal peptide, peptide histidineisoleucine, galanin, substance P, neurokinin A and calcitonin gene-related peptide around blood vessels and follicles. Many of these transmitter candidates were found to co-exist with each other in different combinations in different subpopulations of neurons. Sympathectomy eliminated all noradrenalin- and noradrenalin/neuropeptide Y-containing fibres in the thyroid. Cervical vagotomy eliminated about 50% of the galanin-, substance P- and calcitonin generelated peptide-containing fibres. Local denervation (removal of the thyroid ganglion and the thyroid nerve) eliminated all galanin- and substance P-immunoreactive fibres and the majority of noradrenalin-,noradrenalin/neuropeptide Y-, vasoactive intestinal peptide- and calcitonin gene-related peptide-containing fibres in the thyroid gland. Injection of True Blue into the thyroid gland labelled cell bodies in the thyroid ganglion, the laryngeal ganglion, the superior cervical ganglion, the jugular-nodose ganglionic complex, the dorsal root ganglia (C 2–C 5) and the trigeminal ganglion. Judging from the number of labelled nerve cell bodies, the superior cervical ganglion and the thyroid ganglion contribute most to the thyroid innervation, while the laryngeal ganglion and the trigeminal ganglion contribute least. The True Blue-labelled ganglia were examined for the presence of various populations of nerve cell bodies (only major populations are listed). The thyroid ganglion harboured neuropeptide Y, vasoactive intestinal peptide and galanin/vasoactive intestinal peptide cell bodies (in order of predominance); the laryngeal ganglion galanin/vasoactive intestinal peptide, vasoactive intestinal peptide and calcitonin gene-related peptide cell bodies; the superior cervical ganglion noradenalin/neuropeptide Y and noradrenalin cell bodies; the jugular ganglion calcitonin gene-related peptide, substance P/calcitonin gene-related peptide and galanin/substance P/calcitonin gene-related peptide cell bodies; the nodose ganglion vasoactive intestinal peptide and vasoactive intestinal peptide/galanin cell bodies; the dorsal root ganglia (C 2–C 5) and the trigeminal ganglion calcitonin gene-related peptide, substance P/calcitonin gene-related peptide and galanin/substance P/calcitonin gene-related peptide cell bodies. Results from denervation and tracer experiments indicate that all noradrenalin-containing and the majority of neuropeptide Y-containing nerve fibres in the thyroid derive from the superior cervical ganglion (sympathetic nerve supply). All vasoactive intestinal peptide- and a minor population of neuropeptide Y- and galanin-containing fibres in the thyroid gland originate in the thyroid ganglion (parasympathetic nerve supply). Nerve fibres containing galanin, substance P and/or calcitonin generelated peptide emanate from the jugular, cervical dorsal root and/or trigeminal ganglia (sensory nerve supply). Together the findings indicate that several ganglia project to the thyroid and that many neuropeptides may be involved in the control of thyroid activity.

Distribution of calcitonin gene-related peptide-containing fibers in the urinary bladder of the rat and their origin

By use of indirect immunofluorescence, this study demonstrated the presence of calcitonin gene-related peptide-like immunoreactive (CGRPI) fibers in the bladder of the rat. These fibers were abundant in the muscle layer, in which they ran parallel to the muscles, submucosa, and epithelium. No immunoreactive cells were detected. We also examined the origins of these fibers, using a method that combined biotinized retrograde tracer (biotin-wheat germ agglutinin) (B-WGA) and immunocytochemistry. Injection of the tracer into the bladder resulted in the demonstration of small to medium-sized labeled cells that contain CGRPI structures in single dorsal root ganglion cells mostly at the level of L6 and S1, but also a few at L2. Double-staining for CGRPI and immunoreactive P-like substance (SPI) indicated that there are cells in the dorsal root ganglia at the level of L6 and S1 that react to both, but that there are many CGRPI-positive cells that contain no demonstrable SPI; most of the latter are large.

Calcitonin gene-related peptide in cardiovascular tissues of the rat

The distribution of calcitonin gene-related peptide immunoreactivity in the cardiovascular system of the rat was investigated by radioimmunoassay and immunocytochemistry. The nature of the immunoreactivity was studied by gel permeation and high performance liquid chromatography. Immunocytochemistry demonstrated the existence of calcitonin gene-related peptide-containing nerve fibres throughout the cardiovascular system. These were present in all regions of the heart, particularly in association with the coronary arteries, within the papillary muscles and within the sinoatrial and atrioventricular nodes. Calcitonin gene-related peptide-containing fibres were found mainly in the adventitia of the arteries and veins. Calcitonin gene-related peptide concentrations were high in major arteries and veins but comparatively low in the heart, aortic arch and thoracic aorta. Chromatography showed that approximately 70% of the total immunoreactivity was identical to synthetic calcitonin gene-related peptide. Calcitonin gene-related peptide concentrations in the blood vessels of rats treated neonatally with capsaicin were not found to be significantly different from those in control animals although capsaicin caused significant reductions of calcitonin gene-related peptide levels in certain other tissues. The results of this study suggest that calcitonin gene-related peptide-containing fibres are likely to be of importance in the innervation of vascular tissues and raise the possibility that these fibres are different in character from calcitonin gene-related peptide-containing fibres found in other tissues.