Neuronal galanin is widely distributed in the chicken respiratory tract and coexists with multiple neuropeptides

Summary The mammalian airways are known to be richly innervated by several types of peptide-containing nerve fibers. Galanin-containing fibers are, however, comparatively few. The results of the present immunocytochemical study indicate that the chicken airways receive a notably dense supply of galanin-storing fibers. Other major neuropeptides were neuropeptide Y, vasoactive intestinal peptide and substance P. Nerve fibers containing these peptides were distributed in the trachea, main bronchi, and the lungs. Minor nerve fiber populations contained calcitonin generelated peptide, enkephalin and gastrin-releasing peptide. In the trachea and main bronchi the majority of peptidecontaining nerve fibers was distributed beneath and sometimes also within the epithelium; fibers were fewer in the lamina propria. In the lungs they occurred both in association with the epithelium of small bronchi and in the septa. Adrenergic nerves (using tyrosine hydroxylase as marker) were predominantly distributed in the lamina propria among bundles of smooth muscle and blood vessels. In the nerve fibers associated with the epithelium and in nerve cell bodies in local ganglia of the tracheal wall, galanin was found to coexist with several other neuropeptides (neuropeptide Y, vasoactive intestinal peptide and substance P) suggesting co-expression of multiple neuropeptide genes in the same population of neurons.     

Origin of galanin in nerves of cat airways and colocalization with vasoactive intestinal peptide

Galanin is a 29 amino acid residue neuropeptide. In mammalian airways, galanin is found in nerve fibers associated with airway smooth muscle, bronchial glands, and blood vessels, and in nerve cell bodies of airway ganglia. The present study was conducted to determine if galanin-containing fibers in the walls of feline airways originate from the nerve cell bodies of airway ganglia. The colocalization of galanin with vasoactive intestinal peptide was also investigated. Organotypic cultures of cat airways were held in culture for 0 (nonculture control), 3, 5, and 7 days. After each culture period, the distribution of galanin and the colocalization of galanin with vasoactive intestinal peptide were determined by immunocytochemistry. Galanin-containing fibers were found in bronchial smooth muscle, around bronchial glands and in the walls of bronchial arteries and arterioles throughout the culture period. Nerve fibers and cell bodies containing both galanin and vasoactive intestinal peptide were observed after all culture periods. Nerve fibers and cells bodies that contained galanin frequently contained vasoactive intestinal peptide as well, but nerve fibers with only galanin or vasoactive intestinal were also observed. Galanin- and vasoactive intestinal peptide-containing nerve fibers and cell bodies were both well maintained throughout the culture period. The findings show that galanin-containing nerve fibers associated with bronchial smooth muscle, bronchial glands, and bronchial arteries, originate from nerve cell bodies of intrinsic airway ganglia, and that galanin and vasoactive intestinal peptide are frequently colocalized in these neurons.     

Neuropeptides in guinea pig trachea: Distribution and evidence for the release of CGRP into tracheal lumen

The airways of the guinea pig are richly innervated by peptide-containing nerve fibers. Among the most abundant neuropeptides are calcitonin gene-related peptide (CGRP) and substance P (SP), which are stored in nerve fibers located predominantly within and beneath the epithelium, and vasoactive intestinal peptide (VIP), which is located in fibers running mainly among smooth muscle bundles and seromucous glands. Sensory denervation (capsaicin treatment) of adult guinea pigs caused an almost total disappearance of CGRP- and SP-containing nerve fibers, while the density of VIP-containing nerve fibers located in smooth muscle seemed to increase. In the isolated trachea, perfused luminally, CGRP was found to appear in the intraluminal fluid after exposure to capsaicin but not after electrical vagal stimulation. CGRP concentrations in the tracheal wall did not change significantly. Luminally applied CGRP did not affect smooth muscle tension, measured as intraluminal volume changes.     

Pituitary adenylate cyclase-activating peptide (PACAP), a new vasoactive intestinal peptide (VIP)-like peptide in the respiratory tract

Summary Pituitary adenylate cyclase-activating peptide (PACAP) is a vasoactive intestinal peptide (VIP)-like peptide recently isolated from ovine hypothalami. Nerve fibers displaying PACAP immunoreactivity were found in the respiratory tract of rats, guinea pigs, ferrets, pigs, sheep and squirrel monkeys. A moderate supply of PACAP-immunoreactive fibers was seen in the nasal mucosa of guinea pigs. Few to moderate numbers of PACAP-containing fibers occurred in the tracheo-bronchial wall of rats, guinea pigs, ferrets, pigs, sheep and squirrel monkeys. The fibers were distributed beneath the epithelium, around blood vessels and seromucous glands, and among bundles of smooth muscle. In the lungs, the immunoreactive fibers were observed close to small bronchioli. A few PACAP-immunoreactive nerve cell bodies were seen in the sphenopalatine and otic ganglia of guinea pigs. Simultaneous double immunostaining of the respiratory tract of sheep and ferrets revealed that all PACAP-containing nerve fibers stored VIP. We suggest that neuronal PACAP may take part in the regulation of smooth muscle tone and glandular secretion.     

Catecholamine- and acetylcholinesterase-containing nerves in human lower respiratory tract

Summary The innervation of human lower respiratory tract was studied with special emphasis on airways with sodium-potassium glyoxylic acid (SPG) and acetylcholinesterase (AChE) methods to demonstrate catecholamine-containing and acetylcholinesterase-containing nerve fibers. AChE-method revealed a rich network of cholinesterase positive nerves both inside the bronchial glands where they run around and between the acini, and the airway smooth muscle from secondary bronchi to terminal bronchioli. No AChE-positive fibers were found in connection with the blood vessels or within the epithelium of bronchi or bonchioli. The AChE-positive nerve fibers in bronchial smooth muscle greatly outnumbered those containing catecholamine. The SPG-method revealed the presence of adrenergic nerves from the level of secondary bronchi to that of terminal bronchioli. These nerve fibers were most abundant in bronchial glands, where their amount was equal and distribution similar to those of AChE-containing nerve fibers. Outside the glands adrenergic fibers were constantly seen in connection with the bronchial blood vessels in connective tissues surrounding bronchi. A few nerve fibers were also present in airway smooth muscle from the secondary bronchi to terminal bronchioli.     

Innervation of lower airways and neuropeptide effects on bronchial and vascular tone in the pig

Summary The occurrence and distribution of peptide-containing nerve fibres [substance P (SP), calcitonin gene-related peptide (CGRP), vasoactive intestinal polypeptide (VIP), peptide histidine isoleucine (PHI), neuropeptide Y (NPY)] and noradrenergic nerve fibres [tyrosine hydroxylase (TH)- and dopamine beta hydroxylase (DBH)-positive] in the airways of the pig were studied by means of immunohistochemistry. SP- and CGRP-immunoreactive (-IR) nerve fibres were present close to and within the lining respiratory epithelium, around blood vessels, within the tracheobronchial smooth muscle layer and around local tracheobronchial ganglion cells. The content of CGRP- and neurokinin A (NKA)-like immunoreactivity (-LI) measured by radioimmunoassay (RIA) was twice as high in the trachea compared to that in the peripheral bronchi. SP was a more potent constrictor agent than NKA on pig bronchi in vitro. CGRP had a relaxant effect on precontracted pig bronchi. On blood vessels CGRP exerted a relaxant effect that was more pronounced on pulmonary arteries than on bronchial arteries. VIP/PHI-IR fibres were seen in association with exocrine glands and in the tracheobronchial smooth muscle layer. VIP-positive nerve fibres were abundant around blood vessels in the trachea but sparse or absent around blood vessels in the peripheral bronchi. This histological finding was supported by RIA; it was shown that the content of peptides displaying VIP-like immunoreactivity (-LI) was 18 times higher in the trachea compared to peripheral bronchi. VIP was equally potent as CGRP in relaxing precontracted pig bronchi in vitro. Both bronchial and pulmonary arteries were relaxed by VIP. NPY was colocalized with VIP in tracheal periglandular nerve fibres and in nerve fibres within the tracheobronchial smooth muscle layer. NPY was also present in noradrenergic (DBH-positive) vascular nerve fibres. The content of NPY was much higher (15-fold) in the trachea compared to small bronchi. NPY caused a contraction of both pulmonary and bronchial arteries. The bronchial smooth muscle contraction to field stimulation in vitro was purely cholinergic. A non-cholinergic relaxatory effect following field stimulation was observed after bronchial precontraction. Capsaicin had no effect on pig bronchi in vitro.     

Comparative distribution of neuropeptide tyrosine-,vasoactive intestinal polypeptide-, substance P-immunoreactive,acetylcholinesterase-positive and noradrenergic nerves in the reproductive tract of the female rat

Summary Location, distribution and density of nerve fibers immunoreactive to neuropeptide tyrosine, vasoactive intestinal polypeptide and substance P were studied in the reproductive tract of the female rat and compared with acetylcholinesterase-positive (cholinergic) and noradrenergic nerves. Plexuses of all types of fibers were present in the vagina, uterine cervix, uterine horn and oviduct. In the tubular reproductive organs all of these types of nerve fibers appeared to innervate vascular and non-vascular smooth muscle and nearly all types of fibers formed plexuses subjacent to the epithelium lining the organs. Individual fibers of all classes appeared to innervate fascicles of smooth muscle in the mesometrium of the uterine horn. A few acetylcholinesterase-positive and substance P-immunoreactive fibers were present in the ovary but no vasoactive intestinal polypeptide-immunoreactive nerves were observed. Noradrenergic and neuropeptide tyrosine-immunoreactive nerves were numerous in the ovary where they were seen in the interstitial gland tissue and associated with follicles and blood vessels. It is suggested that these nerves may influence hemodynamic events and non-vascular smooth muscle in such functions as transport of sperm and ova and parturition. Substance P-immunoreactive nerve fibers are likely to be sensory fibers that could have roles in neurohormonal reflexes.     

The distribution and colocalization of neuropeptides and 5-hydroxytryptamine in pelvic nerves supplying the posterior large intestine of the toad,Bufo marinus

The distribution and colocalization of neuropeptides and 5-hydroxytryptamine in the posterior portion of the large intestine of the toad was studied using single- and dual-label immunohistochemistry. Neurons containing colocalized galanin/somatostatin or vasoactive intestinal peptide alone were observed along intramural pelvic nerves. Some of the galanin/somatostatin neurons also contained 5-hydroxytryptamine. Synaptic boutons containing colocalized calcitonin gene-related peptide/vasoactive intestinal peptide were associated with the galanin/somatostatin neurons. The muscle of the large intestine was also innervated by axons containing galamin/somatostatin, vasoactive intestinal peptide/calcitonin gene-related peptide or vasoactive intestinal peptide alone. Nerve fibres containing calcitonin gene-related peptide/substance P, probably representing primary afferent nerves, were also associated with muscle bundles. Submucosal blood vessels carried dense plexuses of fibres containing vasoactive intestinal peptide alone or and calcitonin gene-related peptide/substance P. Adrenergic perivascular nerves also contained galanin and neuropeptide Y.     

Calcitonin gene-related peptide and substance P in the pharynx and lung of the bullfrog,Rana catesbeiana

Indirect double immunofluorescence labelling in the pharynx and lung of the bullfrog, Rana catesbeiana, demonstrated the occurrence, distribution, and coexistence of two neuropeptides. In the pharynx, immunoreactive calcitonin gene-related peptide (CGRP) and substance P (SP) were localized in nerve fibers distributed within and just beneath the ciliated epithelium. In the lung, CGRP and SP were localized in nerve fibers in five principal locations: 1) within the smooth muscle layer in the interfaveolar septa; 2) in the luminal thickened edges of the septa; 3) around the pulmonary vasculature; 4) within, and 5) under the ciliated epithelium. Within the smooth muscle layer in the septa, luminal thickened septa, and around blood vessels, almost all fibers showed coexistence of CGRP and SP. Within and just beneath the ciliated epithelium in the thickened septa, all fibers showed coexistence of CGRP and SP. No immunoreactivity for vasoactive intestinal polypeptide, neuropeptide Y, galanin, somatostatin, FMRFamide, and leucine-and methionine-enkephalins was detected in the nerve fibers within the larynx and the lung. Together with our previous data, the present findings suggest that peptidergic mechanisms are involved in the regulation of amphibian respiratory systems throughout their life.     

Changes in surviving nerve fibers associated with submucosal arteries following extrinsic denervation of the small intestine

Summary The neuropeptide content of nerve fibers associated with submucosal arteries in the small intestine of guinea pigs was studied in whole-mount preparations using immunohistochemical methods. Tissues were obtained from normal animals or animals in which the small intestine had been extrinsically denervated. In normal animals, submucosal arteries are innervated by extrinsic sensory nerve fibers which contain both substance P and calcitonin gene-related peptide, and by sympathetic noradrenergic nerve fibers. In preparations obtained from animals 5–9 days after denervation, nerve fibers which contained substance P without detectable calcitonin gene-related peptide were associated with a few submucosal arteries. Nerve fibers which contained vasoactive intestinal peptide were also associated with some arteries. By 42–48 days after extrinsic denervation, substance P-containing fibers (without calcitonin gene-related peptide) and vasoactive intestinal peptide-containing fibers were associated with nearly every blood vessel. The extrinsic sympathetic nerve fibers did not regenerate during the course of this study. The nerve fibers associated with submucosal arteries in denervated tissues were not sensitive to capsaicin treatment.The alteration in the innervation of submucosal arterioles that follows extrinsic denervation of the gut may reflect either an increase in the neuropeptide content of the fibers, synthesis of a new peptide, or an increase in the number of fibers as a result of axonal sprouting.     

Calcitonin gene-related peptide-like immunoreactivity in nerve fibers in the human skin. Relation to fibers containing substance P-, somatostatin- and vasocactive intestinalpolypeptide-like immunoreactivity

Calcitonin gene-related peptide-like immunoreactivity was demonstrated in in sensory nerve fibers in the epidermis and dermis as free nerve endings and around blood vessels and hair follicles of the human finger pad and arm skin. The vast majority of the calcitonin gene-related immunoreactive fibers was shown to display also substance P-like immunoreactivity and a few fibers in the dermis were somatostatin positive. No fibers displaying both substance P and somatostatin-like immunoreactivity were found but a few substance P immunoreactive fibers in the dermis-epidermis region were found to contain also vasointestinal polypeptide-like immunoreactivity. In the sweat glands, abundant calcitonin gene-related peptide positive, but substance P negative, fibers were observed with a similar distribution pattern as the vasoactive intestinal polypeptide immunoreactive fibers and these fibers were suggested to be of sympathetic origin.     

Calcitonin gene-related peptide-like immunoreactivity in nerve fibers in the human skin

Summary Calcitonin gene-related peptide-like immunoreactivity was demonstrated in in sensory nerve fibers in the epidermis and dermis as free nerve endings and around blood vessels and hair follicles of the human finger pad and arm skin. The vast majority of the calcitonin generelated immunoreactive fibers was shown to display also substance P-like immunoreactivity and a few fibers in the dermis were somatostatin positive. No fibers displaying both substance P and somatostatin-like immunoreactivity were found but a few substance P immunoreactive fibers in the dermis-epidermis region were found to contain also vasointestinal polypeptide-like immunoreactivity. In the sweat glands, abundant calcitonin gene-related peptide positive, but substance P negative, fibers were observed with a similar distribution pattern as the vasoactive intestinal polypeptide immunoreactive fibers and these fibers were suggested to be of sympathetic origin.     

Immunohistochemistry of Grandry corpuscles in the oral mucosa of the duck bill: a light- and electron-microscopic study

Grandry corpuscles in the oral mucosa of the upper bill of the duck were immunohistochemically studied using antisera against calcitonin gene-related peptide (CGRP), galanin, methionine-enkephalin, neuropeptide Y (NPY), somatostatin, substance P (SP) and vasoactive intestinal peptide (VIP). Grandry corpuscles in the lamina propria selectively showed only SP-like immunoreactivity. Herbst corpuscles distributed near Grandry corpuscles were negative to all antisera applied. Although immunoreactive products in the Grandry corpuscles were found as granules in the peripheral cytoplasm of the Grandry cell, the axon terminals and satellite cells exhibited no reactivity. In pre-embedding electron-microscopic sections, SP-like immunoreactive products visualized with 3,3-diaminobezidine were localized in the granules of Grandry cells, but no labeling was observed in the cytoplasmic matrix or cell organelles. Electron-immunocytochemical labeling with colloidal gold by the post-embedding method clearly demonstrated that the SP antigen was localized only in the granules. It is presumed that Grandry cells have a secretory function. However, the function and the method of release of the SP contained in the observed granules remains obscure. Some CGRP-, NPY-, SP- and VIP-like-immunoreactive nerve fibers with varicosities associated with blood vessels and nerve fiber bundles of various sizes were observed in the lamina propria, but no such fibers penetrated into the intraepitherial layer. Nerve fibers positive for SP and VIP were also found in the interlobular connective tissue of the palatine glands. Some SP-positive neurons were detected in the vicinity of the palatine glands.     

Occurrence and distribution of calcitonin gene-related peptide in the mammalian respiratory tract and middle ear

Summary Nerve fibres displaying immunoreactivity to calcitonin gene-related peptide (CGRP) are abundantly distributed in the respiratory tract of man, dog, cat, guineapig, rat and mouse. Numerous fine, beaded CGRP fibres were seen in the middle ear mucosa, and a moderate supply was found in the ear drum. In the nasal mucosa and in the wall of the Eustachian tube CGRP fibres occurred around blood vessels, arteries in particular. A conspiciously rich supply of CGRP fibres was seen beneath and within the epithelium. In addition, a few fibres were seen in smooth muscle bundles and close to sero-mucous glands. In the tracheo-bronchial wall CGRP fibres were distributed beneath and within the epithelium, in vascular and non-vascular smooth muscle and sometimes close to small glands. A few CGRP-immunoreactive endocrine-like cells were, in addition, distributed in the tracheal epithelium of cat, rat and mouse. The trigeminal, spinal and nodose ganglia, studied in rats and guinea-pigs, harboured numerous CGRP-immunoreactive nerve cell bodies. The cervical sympathetic ganglia were devoid of immunoreactive neuronal perikarya. Surgical and chemical (6-hydroxydopamine treatment) sympathectomy did not affect the number and distribution of CGRP fibres. The distribution of CGRP fibres in the respiratory tract suggests that CGRP may take part in sensory transmission. In addition, CGRP may affect the regulation of local blood flow, smooth muscle tone and glandular secretion.     

An immunohistochemical study of the innervation of the large intestine of the toad (Bufo marinus)

The distribution of intrinsic enteric neurons and extrinsic autonomic and sensory neurons in the large intestine of the toad, Bufo marinus, was examined using immunohistochemistry and glyoxylic acid-induced fluoresecence. Three populations of extrinsic nerves were found: unipolar neurons with morphology and location typical of parasympathetic postganglionic neurons containing immunoreactivity to galanin, somatostatin and 5-hydroxytryptamine were present in longitudinally running nerve trunks in the posterior large intestine and projected to the muscle layers and myenteric plexus throughout the large intestine. Sympathetic adrenergic fibres supplied a dense innervation to the circular muscle layer, myenteric plexus and blood vessels. Axons containing colocalized calcitonin gene-related peptide immunoractivity and substance P immunoreactivity distributed to all layers of the large intestine and are thought to be axons of primary afferent neurons. Five populations of enteric neurons were found. These contained immunoreactivity to vasoactive intestinal peptide, which distributed to all layers of the large intestine; galanin/vasoactive intestinal peptide, which projected to the submucosa and mucosa; calcitonin gene-related peptide/vasoactive intestinal peptide, which supplied the circular muscle, submucosa and mucosa; galanin, which projected to the submucosa and mucosa; and enkephalin, which supplied the circular muscle layer.     

Pituitary adenylate cyclase activating peptide (PACAP) in guinea-pig lung: distribution and dilatory effects.

The lower airways of guinea-pigs were analyzed for pituitary adenylate cyclase activating peptide (PACAP) using immunocytochemistry. In the trachea a moderate supply of PACAP-immunoreactive nerve fibers occurred around smooth muscle bundles, glands and small blood vessels. In the lung, PACAP-immunoreactive nerve fibers were distributed around small glands and bronchi. A rich supply of PACAP immunoreactive nerve fibers was found around blood vessels in the lungs. PACAP-suppressed smooth muscle responses were analysed using isolated circular segments of trachea, pulmonary arteries and aorta of guinea-pigs. In both airways and arteries PACAP caused a concentration-dependent relaxation of precontracted segments. The maximal relaxation effects were more pronounced in the airways than in the arteries while the order of potency was aorta greater than pulmonary artery greater than trachea. The effect of PACAP was compared to those of acetylcholine (ACh) and vasoactive intestinal peptide (VIP). In the pulmonary artery the vasomotor responses expressed as maximal dilatation had the order: ACh greater than VIP = PACAP while the order of potency was PACAP = VIP greater than ACh. In the trachea, PACAP was slightly more potent than VIP. The relaxatory responses to PACAP in the trachea and the intrapulmonary arteries were unaffected by pretreatment with atropine, prazosin, yohimbine, propranolol, mepyramine, cimetidine and Spantide. Removal of the endothelium abolished PACAP-induced vascular relaxation. Conceivably, PACAP-containing nerve fibers play a role in the regulation of airway resistance and local blood flow.     

Peptidergic innervation of rat lymphoid tissue and lung: Relation to mast cells and sensitivity to capsaicin and immunization

Summary The peptidergic innervation of lymphoid tissue and the lung in relation to mast cells was studied in rat. The sensitivity of neuropeptide-containing nerves to capsaicin treatment and immunization was also examined. Measurements of the content of neurokinin A and calcitonin gene-related peptide revealed that the lung contained the highest content of both neuropeptides; lymph nodes had intermediate levels, whereas the spleen had the lowest content. Immuhohistochemistry showed that the calcitonin gene-related peptide- and neurokinin A-immunoreactive nerves in lymph nodes were mainly found around blood vessels, whereas in the lung the nerves were present within the lining respiratory epithelium, bronchial smooth muscle, around blood vessels and close to lymphoid aggregates. Combined immunohistochemistry for serotonin (5-hydroxytryptamine), as a marker for mast cells, and tachykinins or calcitonin gene-related peptide revealed that a close association was often present between the nerves and 5-hydroxytryptamine-positive cells in the bronchi of the lung, while 5-hydroxytryptamine-positive cells were not observed in lymph nodes. The neurokinin A and calcitonin gene-related peptide content in lymph nodes, spleen and lung, but not the content of neuropeptide Y, was markedly decreased by capsaicin treatment, suggesting a sensory origin for the two former peptides. Aerosol immunization increased the levels of calcitonin gene-related peptide in the lung, whereas the content in mediastinal lymph nodes was not affected. These data demonstrate a peptidergic innervation mainly of blood vessels in lymphoid tissue and a close relation between sensory nerves and mast cells as well as lymphoid aggregates in the bronchi of the lung. This further suggests that the sensory innervation of lymph nodes is mainly related to regulation of vascular tone and lymph flow. Furthermore, at the site of immunization, i.e., in the airway mucosa, sensory nerve mediators may interact both with mast cells and lymphoid cells.     

Immunohistochemical characteristics of nerve fibres supplying the porcine vas deferens. A colocalisation study

 Double-labelling immunofluorescence was used to investigate the coexistence of the catecholamine-synthesising enzymes, tyrosine hydroxylase and dopamine-β-hydroxylase and several neuropeptides including neuropeptide Y, vasoactive intestinal polypeptide, Leu⁵-enkephalin, somatostatin, calcitonin gene-related peptide and substance P in nerve fibres supplying the vas deferens in juvenile and adult pigs. The study has revealed three major populations of nerve terminals innervating the organ: (1) noradrenergic fibres; (2) non-noradrenergic (putative cholinergic) fibres containing vasoactive intestinal polypeptide, neuropeptide Y and somatostatin, supplying almost exclusively the lamina propria; and (3) non-noradrenergic, presumably sensory fibres, containing calcitonin gene-related peptide and substance P. The population of noradrenergic nerves can be divided into three subpopulations: a somatostatin-containing, a Leu⁵-enkephalin-containing and a subpopulation immunonegative to the peptides investigated, in descending order of magnitude. Coexistence patterns of the substances existing within nerve fibres supplying the vas deferens blood vessels are clearly different from those found in nerve fibres innervating the organ wall. The majority of the noradrenergic fibres associated with blood vessels contain neuropeptide Y only, while non-noradrenergic perivascular nerves contain predominantly vasoactive intestinal polypeptide. The possibility of different sources of origin of the particular nerve fibre subpopulations supplying the porcine vas deferens and its blood vessels is discussed. Accepted: 23 October 1996     

Calcitonin gene-related peptide (CGRP): perivascular distribution and vasodilatory effects

The distribution of perivascular nerve fibers displaying calcitonin gene-related peptide (CGRP) immunoreactivity and the effect of CGRP on vascular smooth muscle were studied in the guinea-pig. Perivascular CGRP fibers were seen in all vascular beds. Generally, they were more numerous around arteries than veins. Small arteries in the respiratory tract, gastrointestinal tract and genitourinary tract had numerous CGRP fibers. The gastroepiploic artery in particular received a rich supply of such fibers. Coronary blood vessels had a moderate supply of CGRP fibers. In the heart, a moderate number of CGRP fibers was seen running close to myocardial fibers. The atria had a richer supply than the ventricles. Numerous CGRP immunoreactive nerve cell bodies and nerve fibers were seen in sensory (trigeminal, jugular and spinal dorsal root) ganglia. Sequential or double immunostaining with antibodies against substance P and CGRP suggested co-existence of the two peptides in nerve cell bodies in the ganglia and in perivascular fibers. In agreement with previous findings CGRP turned out to be a strong vasodilator in vitro as tested on several blood vessels (e.g. basilar, gastroepiploic and mesenteric arteries). Conceivably, perivascular CGRP/SP fibers have a dual role as regulator of local blood flow and as carrier of sensory information.     

Nonvagal origin of galanin-containing nerve terminals innervating striated muscle fibers of the rat esophagus

We investigated the origin of galanin-positive nerve fibers on motor endplates in rat esophagus using anterograde 1,1′-dioleyl-3,3,3′,3′-tetramethylindocarbocyanine methane sulfonate (DiI) tracing from the nucleus ambiguus combined with galanin immunocytochemistry and calcitonin gene-related peptide immunocytochemistry. To demonstrate spatial relationships of galanin-positive nerve fibers to vagal and enteric nerve fibers on motor endplates, we combined galanin immunocytochemistry with calcitonin gene-related peptide immunostaining for labeling of vagal terminals, and vasoactive intestinal peptide immunoreactivity and NADPH-diaphorase histochemistry for demonstration of enteric nerve fibers. Within fine varicose nerve fibers, galanin was colocalized with vasoactive intestinal peptide and NADPH-diaphorase to a high degree and turned out to be completely separated from calcitonin gene-related peptide-positive or anterogradely DiI-labeled vagal motor terminals. These results indicate that the enteric nervous system is the most important and possibly the only source of galanin-positive nerve terminals on motor endplates in rat esophagus. Galanin may be, in addition to nitric oxide and vasoactive intestinal peptide, a mediator of the enteric coinnervation of striated muscle in this organ.