Colocalization of neuropeptides and NADPH-diaphorase in the intra-adrenal neuronal cell bodies and fibres of the rat

Colocalization of vasoactive intestinal peptide, neuropeptide Y, calcitonin gene-related peptide, substance P, and tyrosine hydroxylase, respectively, with NADPH-diaphorase staining in rat adrenal gland was investigated using the double labelling technique. All vasoactive intestinal peptide- and some neuropeptide Y-immunoreactive intrinsic neuronal cell bodies seen in the gland were double stained with NADPH-diaphorase. Double labelling also occurred in some nerve fibres immunoreactive to vasoactive intestinal peptide and neuropeptide Y in the medulla and cortex. No colocalization of calcitonin gene-related peptide, substance P or tyrosine hydroxylase immunoreactivity with NADPH-diaphorase staining was observed. However, nerve fibres with varicosities immunoreactive for all the neuropeptides examined were closely associated with some of the NADPH-diaphorase-stained neuronal cell bodies. Thus, in rat adrenal gland, nitric oxide is synthesized in all ganglion cells containing vasoactive intestinal peptide and in some containing neuropeptide Y, but not in those containing calcitonin gene-related peptide, substance P or tyrosine hydroxylase.     

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.     

Colocalization of NADPH-diaphorase activity and certain neuropeptides in the esophagus of opossum (Didelphis virginiana)

Nitric oxide and various neuropeptides in the myenteric plexus regulate esophageal motility. We sought colocalization of nitric oxide synthase and neuropeptides in frozen sections of mid-portion of smoothmuscled opossum esophagus using NADPH-diaphorase activity to mark the synthase and immunoreactivity to detect peptides. The peptides, all with demonstrated physiological activity in this organ, were calcitonin generelated peptide, galanin, neuropeptide Y, substance P, and vasoactive intestinal polypeptide. The ExtrAvidin Peroxidase immunostain for each peptide was carried up to the final peroxidase reaction with 3-amino-9-ethylcarbazole. The NADPH-diaphorase reaction was applied with short incubation to provide light staining just before the peroxidase reaction was performed. We examined sections for the proportions of singly and dually labeled nerve cells in the myenteric plexus. NADPH-diaphorase activity was highly colocalized with calcitonin gene-related peptide (59%), galanin (54%), and vasoactive intestinal polypeptide (53%). It showed little colocalization with neuropeptide Y (10%) and substance P (8%). The proportions of all nerve cells containing each of the substances were: NADPH-diaphorase-33%, calcitonin gene-related peptide-30%, galanin-55%, neuropeptide Y-16%, substance P-35%, and vasoactive intestinal polypeptide-58%. We conclude that the nerves responsible for peristalsis in the esophagus may act by releasing nitric oxide along with other inhibitory substances, calcitonin gene-related peptide, galanin, and vasoactive intestinal polypeptide, but not excitatory substances, neuropeptide Y and substance P.     

Galanin-immunoreactive nerves in the rat iris: alterations induced by denervations

Summary The iris and choroid membrane of the adult rat contain nerve fibers expressing immunoreactivity to the neuropeptide galanin. The density and distribution of galanin-positive nerve fibers varied from iris to iris and, particularly, among animals. Smooth, non-terminal axons were seen running in nerve bundles consisting of otherwise negative fibers. From the choroid membrane these bundles reached the iris via the ciliary body. Axons were frequently seen to branch giving rise to a sparse system of varicose, single fibers in the dilator plate and sphincter area. Galanin-positive fibers were sometimes also seen outlining blood vessels.Capsaicin, in a dose that causes permanent depletion of substance P- and cholecystokinin-immunoreactive fibers in the iris, caused no change in amount of galanin-positive fibers. Removal of the superior cervical ganglion caused a rapid and pronounced increase in the number of galanin-immunoreactive nerve fibers. Similarly, removal of the ciliary ganglion appeared to increase galanin immunoreactivity, while removal of the pterygopalatine ganglion was less effective. Lesioning of the trigeminal ganglion caused a disappearance of galanin immunoreactivity. The sympathetectomy-induced increase was counteracted by capsaicin.Galanin-positive nerve cell bodies were present in both the superior cervical and the trigeminal ganglia. In the superior cervical ganglion, immunoreactive galanin did not seem to coexist with neuropeptide Y-positive cells; in the trigeminal ganglion, some galanin-positive cells also contained calcitonin gene-related peptide (CGRP) immunoreactivity, while most cells did not. In the iris, double-staining suggested that CGRP and galanin immunoreactivities were contained in different fiber populations.We conclude that the rat iris and choroid membrane contain a sparse plexus of nerve fibers expressing galanin-like immunoreactivity. It is suggested that these fibers are derived from the trigeminal ganglion. The iris is able to respond with a pronounced increase in number of galanin-immunoreactive nerve fibers to certain denervation procedures.     

Studies on the distribution of calcitonin gene-related peptide-like and substance P-like immunoreactivities in rat hind limb muscles

Summary The tibialis anterior, extensor digitorum longus and soleus muscles in the rat were examined with respect to the presence of calcitonin gene-related peptide-like as well as substance P-like immunoreactivity. In some of the motor endplates in these muscles, identified by staining for acetylcholinesterase activity, calcitonin gene-related peptide-like immunoreactivity was detected, but in others it was not. Calcitonin gene-related peptide-like immunoreactivity was found to coexist with substance-P-like immunoreactivity in nerve fibres located outside and inside the capsule of the muscle spindles, as well as in nerve fibres located in nerve fascicles. These fibres presumably represent sensory nerve fibres. Calcitonin gene-related peptide-like immunoreactivity, but not substance P-like immunoreactivity, was also detected, in cap-like structures located on the surface of the intrafusal muscle fibres in the polar regions of the spindles, structures which are likely to correspond to motor plate endings. The observations suggest that calcitonin gene-related peptide is heterogeneously present in the endplates of rat hind limb muscles, and gives for the first time immunohistochemical evidence for the presence of calcitonin gene-related peptide and substance P in the innervation of muscle spindles.     

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

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

Localization of immunoreactive neuropeptides in the kidney of the bullfrog,Rana catesbeiana,by immunofluorescence

Indirect double immunofluorescence labelling for demonstrating nine neuropeptides in the kidney of the bullfrog, Rana catesbeiana, revealed for the first time the occurrence, distribution, and coexistence of certain neuropeptides in the kidney of the submammalian vertebrates. Substance P, neuropeptide Y, and calcitonin generelated peptide were localized in nerve fibers distributed along the afferent arterioles connected with the glomeruli, and along the capillary network between uriniferous tubules. Neuropeptide Y and calcitonin gene-related peptide immunoreactive fibers were more numerous than substance P immunoreactive fibers. In these two regions, about one half of the neuropeptide Y or calcitonin in gene-related peptide fibers contained substance P. No immunoreactivity of vasoactive intestinal polypeptide, somatostatin, FMRFamide, or leucine- and methionine-enkephalins was detected in the bullfrog kidney.     

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.     

Distribution of peptidergic nerve fibres in bullfrog lingual papillae demonstrated by a combination of double immunofluorescence labelling and a multiple dye filter

Summary Immunoreactivity of substance P, calcitonin gene-related peptide, vasoactive intestinal polypeptide, neuropeptide Y, and galanin is localized in nerve fibres distributed in the fungiform and filiform papillae of the tongue of the bullfrog,Rana catesbeiana. A combination of indirect double immunofluorescence labelling and a multiple dye filter system clearly demonstrated that all substance P fibres in the connective tissue core of the fungiform and filiform papillae, and within the rim of ciliated cells located on the top of the fungiform papillae showed coexistence with calcitonin gene-related peptide. A few fibres in the epithelial discs, which are located in the centre of the top of the fungiform papillae, showed the immunoreactivity of calcitonin gene-related peptide alone. There were no substance P fibres which showed coexistence with vasoactive intestinal polypeptide, galanin, and neuropeptide Y. In high magnification images, substance P and vasoactive intestinal polypeptide, and substance P and galanin fibres were recognized as two interwined fibres within the same thin nerve bundle. No immunoreactivity of leucine- and methionine-enkephalins can be detected. These findings suggest that the chemoreceptor function of the bullfrog gustatory organ may be under the control of complicated peptidergic innervation.     

The distribution and colocalization of neuropeptides in perivascular nerves innervating the large arteries and veins of the snake,Elaphe obsoleta

Summary Single- and dual-labelling immunohistochemistry were used to determine the distribution and coexistence of neuropeptides in perivascular nerves of the large arteries and veins of the snake, Elaphe obsoleta, using antibodies for vasoactive intestinal polypeptide, substance P, calcitonin gene-related peptide, neuropeptide Y, galanin, somatostatin, and leu-enkephalin. Blood vessels were sampled from four regions along the body of the snake: region 1, arteries and veins anterior to the heart; region 2, central vasculature 5 cm anterior and 10 cm posterior to the heart; region 3, arteries and veins in a 30-cm region posterior to the liver; and region 4, dorsal aorta and renal arteries, renal and intestinal veins, 5–30 cm cephalad of the vent. A moderate to dense distribution of vasoactive intestinal polypeptide-like immunoreactive fibres was found in most arteries and veins of regions 1–3, but fibres were absent from the vessels of region 4. The majority of vasoactive intestinal polypeptide-like immunoreactive fibres contained colocalized substance P-like immunoreactivity, and these fibres were unaffected by either capsaicin or 6-hydroxydopamine (6-OHDA) pretreatment. In the anterior section of the snake, the vagal trunks contained many cell bodies with colocalized vasoactive intestinal polypeptide and substance P-like immunoreactivity. It is suggested that the vasoactive intestinal polypeptide/substance P-like immunoreactive cell bodies and fibres are parasympathetic postganglionic nerves. Neuropeptide Y-like immunoreactive fibres were observed in all arteries and veins, being most dense in regions 3 and 4. The majority of these fibres also contained colocalized galanin-like immunoreactivity, and were absent in tissues from 6-OHDA pretreated snakes, suggesting that neuropeptide Y and galanin are colocalized in adrenergic nerves. A small number of neuropeptide Y-like immunoreactive fibres contained vasoactive intestinal polypeptide but not galanin, and were unaffected by 6-OHDA treatment. All calcitonin gene-related peptide-like immunoreactive fibres contained colocalized substance P-like immunoreactivity, and these fibres were observed in all vessels, being particularly dense in the carotid artery and jugular veins. All calcitonin gene-related peptide/substance P-like immunoreactive fibres appeared damaged after capsaicin treatment suggesting they represent fibres from afferent sensory neurons. A sparse plexus of somatostatin-like immunoreactive fibres was observed in the vessels only from region 4. No enkephalin-like immunoreactive fibres were found in any blood vessels from any region. This study provides morphological evidence to suggest that there is considerable functional specialization within the components of the rat snake peripheral autonomic system controlling the circulation, in particular the regulation of venous capacitance.     

Distribution of galanin-immunoreactive nerve fibers in the carotid labyrinth of the bullfrog,Rana catesbeiana: comparison with substance P-immunoreactive fibers

Immunoreactivity of galanin (GAL) was detected in the nerve fibers distributed within the intervascular stroma of the bullfrog carotid labyrinth. GAL-immunoreactive fibers are numerous, and some are close to the sinusoidal plexus. Most GAL fibers appear as thin processes with some varicosities. A combination of indirect double immunofluorescence labelling and image processing clearly demonstrated that the distribution patern of GAL fibers is different from that of SP fibers. This indicates that GAL and SP do not coexist in the same nerve fibers. The role of GAL fibers may be different from that of previously reported neuropeptides (substance P, calcitonin gene-related peptide, vasoactive intestinal polypeptide, neuropeptide Y, and others) as a neuromodulator in controlling vascular tone of the labyrinth.     

Immunohistochemical and ultrastructural localisation of peptide-containing nerves and myocardial cells in the human atrial appendage

Summary The innervation and myocardial cells of the human atrial appendage were investigated by means of immunocytochemical and ultrastructural techniques using both tissue sections and whole mount preparations. A dense innervation of the myocardium, blood vessels and endocardium was revealed with antisera to general neuronal (protein gene product 9.5 and synaptophysin) and Schwann cell markers (S-100). The majority of nerve fibres possessed neuropeptide Y immunoreactivity and were found associated with myocardial cells, around small arteries and arterioles at the adventitial-medial border and forming a plexus in the endocardium. Subpopulations of nerve fibres displayed immunoreactivity for vasoactive intestinal polypeptide, somatostatin, substance P and calcitonin gene-related peptide. In whole-mount preparations of endocardium, substance P and calcitonin gene-related peptide immunoreactivities were found to coexist in the same varicose nerve terminals. Ultrastructural studies revealed the presence of numerous varicose terminals associated with myocardial, vascular smooth muscle and endothelial cells. Neuropeptide Y immunoreactivity was localised to large electron-dense secretory vesicles in nerve terminals which also contained numerous small vesicles. Atrial natriuretic peptide immunoreactivity occurred exclusively in myocardial cells where it was localised to large secretory vesicles. The human atrial appendage comprises a neuroendocrine complex of peptidecontaining nerves and myocardial cells producing ANP.     

Distribution of five peptides, three general neuroendocrine markers, and two synaptic-vesicle-associated proteins in the spinal cord and dorsal root ganglia of the adult and newborn dog: an immunocytochemical study

This study describes the immunocytochemical distribution of five neuropeptides (calcitonin gene-related peptide [CGRP], enkephalin, galanin, somatostatin, and substance P), three neuronal markers (neurofilament triplet proteins, neuron-specific enolase [NSE], and protein gene product 9.5), and two synaptic-vesicle-associated proteins (synapsin I and synaptophysin) in the spinal cord and dorsal root ganglia of adult and newborn dogs. CGRP and substance P were the only peptides detectable at birth in the spinal cord; they were present within a small number of immunoreactive fibers concentrated in laminae I-II. CGRP immunoreactivity was also observed in motoneurons and in dorsal root ganglion cells. In adult animals, all peptides under study were localized to varicose fibers forming rich plexuses within laminae I-III and, to a lesser extent, lamina X and the intermediolateral cell columns. Some dorsal root ganglion neurons were CGRP- and/or substance P-immunoreactive. The other antigens were present in the spinal cord and dorsal root ganglia of both adult and newborn animals, with the exception of NSE, which, at birth, was not detectable in spinal cord neurons. Moreover, synapsin I/synaptophysin immunoreactivity, at birth, was restricted to laminae I-II, while in adult dogs, immunostaining was observed in terminal-like elements throughout the spinal neuropil. These results suggest that in the dog spinal cord and dorsal root ganglia, peptide-containing pathways complete their development during postnatal life, together with the full expression of NSE and synapsin I/synaptophysin immunoreactivities. In adulthood, peptide distribution is similar to that described in other mammals, although a relative absence of immunoreactive cell bodies was observed in the spinal cord.     

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.     

Immunohistochemical analysis of intracardiac ganglia of the rat heart

The neurochemistry of intracardiac neurons in whole-mount preparations of the intrinsic ganglia was investigated. This technique allowed the study of the morphology of the ganglionated nerve plexus found within the atria as well as of individual neurons. Intracardiac ganglia formed a ring-like plexus around the entry of the pulmonary veins and were interconnected by a series of fine nerve fibres. All intracardiac neurons contained immunoreactivity to PGP-9.5, choline acetyl transferase (ChAT) and neuropeptide Y (NPY). Two smaller subpopulations were immunoreactive to calbindin or nitric oxide synthase. Furthermore, a subpopulation (approximately 6%) of PGP-9.5/ChAT/NPY-immunoreactive cells lacking both calbindin and nitric oxide synthase (NOS) was surrounded by pericellular baskets immunoreactive to ChAT and calbindin. Vasoactive intestinal peptide (VIP), calcitonin gene-related peptide (CGRP), pituitary adenylate cyclase-activated peptide (PACAP), substance P and tyrosine hydroxylase (TH) immunoreactivity was observed in nerve fibres within the ganglion, but never in neuronal somata. Furthermore, immunoreactivity for NPY was not observed in pericellular baskets surrounding intracardiac neurons, despite being present in all intrinsic neuronal cell bodies. Taken together, the results of this study indicate a moderate level of chemical diversity within the intracardiac neurons of the rat. Such chemical diversity may reflect functional specialisation of neurons in the intracardiac ganglia.This work was supported by a grant-in-aid (G00M0670) from the National Heart Foundation of Australia     

Calcitonin gene-related peptide neurons innervating the canine digestive system.

The pattern of nerve cells and fibers containing calcitonin gene-related peptide immunoreactivity (CGRP-IR) was investigated in the canine digestive tract by means of immunohistochemistry. CGRP-IR nerve fibers innervate all the layers of the gut, including the vasculature, with different densities depending on the region. CGRP-IR processes are sparse in the esophagus and stomach, where they are mostly confined to the enteric plexuses and vasculature. CGRP-IR fibers are quite abundant in the small and large intestine, where they form dense arborizations in the mucosa, and are numerous in the muscularis mucosae, deep muscular plexus and circular muscle. The myenteric and submucous plexuses of the intestine contain dense networks of CGRP-IR fibers and numerous CGRP-IR ganglion cells. On the other hand, in the enteric ganglia of the esophagus and stomach, in the intrapancreatic ganglia and in the ganglionated plexus of the gallbladder, CGRP-IR is restricted to non-varicose processes. A moderate density of CGRP-IR fibers supplies the endocrine and exocrine pancreas, and the fibromuscular layer and lamina propria of the gallbladder. The density of CGRP innervation in different regions can be summarized as follows: intestine > pancreas and gallbladder > or = antrum > cardia > gastric corpus and distal esophagus. CGRP- and tachykinin (TK)-IRs are colocalized in a substantial population of fibers, particularly those distributed to the mucosa, muscularis mucosae and vasculature, whereas there was no evidence of colocalization in intrinsic ganglion cells. The present results suggest that (1) the CGRP innervation of the dog digestive system includes an intrinsic and an extrinsic component, and (2) CGRP- and TK-IRs are co-expressed in extrinsic nerve fibers. These findings extend previous observations in rats and guinea pigs and provide insights into the sites of action of CGRP in the digestive system of the dog, which has served as a model for CGRP functional studies.     

Co-localization of nitric oxide synthase and vasoactive intestinal peptide immunoreactivity in neurons of the major pelvic ganglion projecting to the rat rectum and penis

We demonstrate the existence of nerve fibers possessing substance P (SP) and calcitonin gene-related peptide (CGRP) immunoreactivity in the mouse cervical ventral roots. The distribution of the SP and CGRP fibers was similar, but CGRP fibers were generally more numerous. Both types entered the ventral pia mater or formed hairpin loops, but they did not enter the spinal cord directly through these roots. SP and CGRP fibers in the ventral roots were thin and had many varicosities. We suggest that these SP and CGRP fibers are involved not only in a sensory mechanism, but also in other functions, via the release of SP and CGRP from varicosities in the ventral roots.     

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.     

Combined retrograde tracing and immunohistochemistry of trigeminal ganglion neurons projecting to gingiva or tooth pulps in the lower jaw of the cichlid Tilapia mariae

Rat trigeminal ganglion neurons projecting to the oral mucosa or to tooth pulps have different cell diameters and contain different chemical markers. In the present paper we examine whether trigeminal ganglion neurons sending axons to gingiva or tooth pulps in the lower jaw of the cichlid Tilapia mariae differ in a similar way. Retrograde tracing with fluorescent latex microspheres revealed labelled gingival and pulpal neurons in the caudal part of the trigeminal ganglion. The gingival neurons had a unimodal size distribution (peak 11 μm; range 8–14 μm) and the pulpal neurons exhibited a bimodal size distribution (peaks 12 and 25 μm; range 10–40 μm). Immunohistochemistry revealed a calcitonin gene-related peptide-like immunoreactivity in some 40% of the gingival neurons and a substance P-like immunoreactivity in 30%. Of the small pulpal neurons about 60% exhibited a calcitonin gene-related peptide-like immunoreactivity and 15% showed a substance P-like immunoreactivity. Of the large pulpal neurons some 70% exhibited a calcitonin gene-related peptide-like immunoreactivity. These neurons did not show a substance P-like immunoreactivity. In some animals a few trigeminal ganglion neurons showed a neuropeptide Y- or a vasoactive intestinal polypeptide-like immunoreactivity. Perikarya with a tyrosine hydroxylase- or a choline acetyl transferase-like immunoreactivity were not observed. We conclude that gingiva and tooth pulps in the lower jaw of T. mariae are innervated by trigeminal ganglion neurons, the cell diameters and neuropeptide contents of which differ in a pattern similar to that in the rat. Hence, this seems to represent a conserved evolutionary pattern.