The distribution and origin of nerve fibers immunoreactive for substance P and neurokinin A in the anterior buccal gland of the rat

The distribution and origin of substance P (SP) and neurokinin A (NKA) were studied in rat in the anterior buccal glands, which are minor mucous salivary glands. Indirect immunofluorescence staining showed moderate SP and NKA innervation of salivary acini and interlobular ducts, whereas blood vessels were more sparsely innervated, and there were few nerve fibers in the stroma and around the intralobular ducts. About 10%–20% of the trigeminal ganglion cells showed equally strong immunoreactivity to both SP and NKA. Unilateral denervation of the branches of the trigeminal nerve caused complete disappearance of the stromal fibers and greatly reduced the number of all other SP-immunoreactive and NKA-immunoreactive nerve fibers. In the superior cervical ganglia, SP and NKA immunoreactivity was restricted to small intensely fluorescent cells; SP and NKA immunoreactivity was absent from principal ganglionic cells, and thus sympathectomy had no any effect on the number or distribution of fibers immunoreactive for SP and NKA in the anterior buccal glands. The fibers remaining after sensory denervation could have been of parasympathetic origin, indicating a dual origin of nerves immunoreactive for SP and NKA in these glands. The present data demonstrate that the major part of the glandular SP and NKA innervation in the minor salivary glands derives from the trigeminal ganglia. The distribution of the peripheral nerve fibers indicates that they may play a role in the delivery of potent neuropeptides involved in the vascular, secretory, and motor (myoepithelial cells) functions of salivary glands.     

The sympathetic and parasympathetic nature of neuropeptide Y-immunoreactive nerve fibres in the major salivary glands of the rat

Summary The distribution and origin of neuropeptide Y in the major salivary glands of the rat was studied by indirect immunofluorescence technique. Numerous nerve fibres immunoreactive for the peptide were seen in the parotid and sublingual glands. Most of the fibres were located around blood vessels and salivary acini. In the submandibular gland the number of immunoreactive nerve fibres around the acini was lower in comparison with that in the parotid and sublingual glands. Some immunoreactive nerve fibres were also found around or along intra- and interlobular ducts in all major salivary glands.A large number of the neuropeptide-containing neuronal cell bodies and nerve fibres were detected in the sympathetic superior cervical ganglion. Sympathetic postganglionic nerve trunks of this ganglion contained numerous immunoreactive nerve fibres as well. A subpopulation of the neuronal cell bodies in the submandibular ganglion were immunoreactive to neuropeptide Y.Both uni- and bilateral superior cervical ganglionectomies caused a significant decrease in the number of immunoreactive nerve fibres around the blood vessels in all the major salivary glands. However, these denervations did not affect the density of nerve fibres around the acini and ducts. On the contrary, unilateral parasympathetic denervation by sectioning the auriculotemporal nerve reduced the fibres around the secretory acini in the parotid gland remarkably, while only a minor reduction in the density of immunoreactive fibres associated with the blood vessels of the gland was detected. Unilateral electrocoagulation of the trigeminal nerve branches caused no detectable change in the density of immunoreactive nerve fibres in any of the major salivary glands.On the basis of the present findings it is concluded that neuropeptide Y-reactive nerve fibres present in all major salivary glands around the blood vessels seem to be mainly sympathetic, whereas those around the acini and ducts seems to be of parasympathetic origin.     

Nitric oxide synthase immunoreactive nerves in rat and ferret salivary glands, and effects of denervation

Nitric oxide has been implicated in mechanisms mediating nerve-evoked vasodilatory and secretory responses in salivary glands. In the present study, the occurrence and distribution of nitric oxide synthase (NOS)-immunoreactive nerves in ferret and rat salivary glands were investigated using immunocytochemistry with rabbit and sheep NOS antisera, and using NADPH-diaphorase enzyme histochemistry. In the parotid, submandibular and sublingual glands of the rat and the ferret, NOS-immunoreactive varicose terminals encircled acini and arteries of various sizes. In the ferret, collecting ducts were also supplied with NOS-immunoreactive fibres. In the rat, only the granular ducts of the submandibular gland were supplied with such fibres. The NOS-immunoreactive innervation of acinar cells was more abundant in the rat than in the ferret, whereas the opposite was true for the innervation of blood vessels. No NOS immunoreactivity was observed in the vascular endothelium. In both species, NOS-positive ganglionic cell bodies were found in the hilar regions of the submandibular and sublingual glands, whereas none could be detected in the parotid glands. NADPH-diaphorase reactivity had the same neuronal distribution as NOS immunoreactivity and, in addition, NADPH-diaphorase reactivity was expressed in ductal epithelium. Neither sympathetic denervation (by removal of the superior cervical ganglion) nor treatment with the sensory neurotoxin capsaicin reduced the NOS-immunoreactive innervation of the parotid gland. However, parasympathetic denervation (by cutting the auriculo-temporal nerve) caused an almost total disappearance of the NOS-immunoreactive innervation. The present findings provide a morphological background to the suggested role of nitric oxide in parasympathetic secretory and vascular responses of salivary glands. This revised version was published online in November 2006 with corrections to the Cover Date.     

Met5-enkephalin-Arg6-Gly7-Leu8-immunoreactive nerve fibers in the major salivary glands of the rat: evidence for both sympathetic and parasympathetic origin

Summary The localization of the proenkephalin A-derived octapeptide, Met⁵-enkephalin-Arg⁶-Gly⁷-Leu⁸ (MEAGL), was studied in the major salivary glands of Sprague-Dawley and Wistar rats with the indirect immunofluorescence method. MEAGL-immunoreactive nerve fibers were found around the acini, along intra-and interlobular salivary ducts and in close contact with blood vessels. In the parotid and submandibular glands tyrosine hydroxylase (TH) immunoreactivity was observed in nerve fibers around the acini, in association with intra- and interlobular salivary ducts and around blood vessels, while in the sublingual gland TH-immunoreactive nerve fibers were only seen around blood vessels. Parasympathetic neurons in submandibular ganglia contained MEAGL immunoreactivity. Moderate TH immunoreactivity was seen in some neurons of the submandibular ganglia. A subpopulation of sympathetic principal neurons in the superior cervical ganglion were immunoreactive for both MEAGL and TH. In the trigeminal ganglion, no MEAGL-immunoreactive sensory neurons or nerve fibers were observed. Superior cervical ganglionectomies resulted in a complete disappearance of TH-immunoreactive nerve fibers, while MEAGL-immunoreative nerve fibers were still present in the glands. The presence of MEAGL immunoreactivity in neurons of both sympathetic superior cervical ganglia and parasympathetic submandibular ganglia and the results of superior cervical ganglionectomies suggest, that MEAGL-immunoreactive nerve fibers in the major salivary glands of the rat have both sympathetic and parasympathetic origin.     

Effects of surgical denervations on the autonomic nerves in parotid glands of dogs

Summary The innervation of the dog's parotid has been studied by cholinesterase staining and catecholamine fluorescence. In normal glands cholinergic and adrenergic nerves are plentiful around acini, muscular blood vessels, and to a lesser extent striated ducts. The main ducts, although surrounded by many cholinesterase-positive nerves, are associated with few adrenergic nerves. Severance of the classical parasympathetic post-ganglionic nerve to the gland, the auriculo-temporal, caused a moderate loss of cholinesterase-positive nerves. When this procedure was combined with section of the nerves on the internal maxillary artery there was a greater loss. Fewest cholinesterase-positive nerves remained when, in addition to these two procedures, the facial nerve was cut. These findings support the concept that all three sets of nerves contain some post-ganglionic parasympathetic fibres for the dog's parotid. The source of the remaining nerves is unknown. Preganglionic parasympathetic denervation by section of the tympanic branch of the glossopharyngeal nerve did not reduce the number of cholinesterase-positive nerves. None of these parasympathetic denervations caused reduction of adrenergic nerves, indicating that they do not travel to the gland with the parasympathetic nerves. After superior cervical ganglionectomy a few scattered fluorescent nerves remained in the gland; their origin is unknown.     

Vasoactive intestinal peptide and substance P in salivary glands of the rat following denervation or duct ligation

Immunoreactive vasoactive intestinal peptide (VIP) and substance P (SP) were studied in parotid, submaxillary and sublingual glands of the rat. The concentration of VIP was highest in the submaxillary gland and lowest in the parotid gland. The concentration of SP was highest in the parotid gland; it was at, or below the limit of detection in the sublingual gland. In the parotid gland the total amounts of VIP and SP were reduced by 95% after parasympathetic denervation (section of the auriculo-temporal nerve). In the submaxillary gland the total amounts of the peptides were unchanged after parasympathetic decentralization (section of the chorda-lingual nerve). In this gland the total amount of SP was reduced by 92% and that of VIP by 50%, when the chorda tympani nerve fibres were cut deep into the hilum. Cutting the nerve fibres at the hilum left the total amounts of the peptides unchanged in the submaxillary gland, whereas in the sublingual gland the total amount of VIP was reduced by 70%. Sympathetic denervation did not reduce the total amounts of the peptides. Duct ligation caused gland atrophy. In the parotid gland the total amounts of VIP and SP were reduced by 40%. In the submaxillary gland the same percentage reduction occurred with regard to SP; however, the total amount of VIP was reduced by 99%. The VIP- and SP-containing nerve fibres reach the salivary glands by the parasympathetic nerves. In both submaxillary and sublingual glands a certain fraction of VIP originates within the glands.     

Production and characterization of a monoclonal antibody against human calcitonin gene-related peptide (CGRP) and its immunohistochemical application to salivary glands

Summary A monoclonal antibody (mAb), 129CD8 was raised against a C-terminal fragment (aa28–37) of -human calcitonin gene-related peptide (CGRP) coupled to bovine serum albumin. The specificity of the monoclonal antibody 129CD8 was corroborated by dot immunobinding experiments, enzyme-linked immunoassay and immunostaining of tissue sections. In vitro studies showed that the mAb 129CD8 readily recognized the fragment 28–37 of -human CGRP and to a slightly lesser degree whole -human CGRP and the fragments containing the C-terminal part of the molecule. The mAb 129CD8 also recognized the -human CGRP but not the -rat CGRP. The mAb 129CD8 did not react with substance P, katacalcin, calcitonin, amylin or fragments of -human CGRP lacking the C-terminal part of the molecule.Immunocytochemical staining was performed on human skin, guinea-pig thyroid and salivary glands and the trigeminal ganglion, and rat thyroid gland. Our findings demonstrate, in keeping with previous studies, that in human skin, nerve fibres containing CGRP immunoreactivity are found in both epidermis and dermis. In accordance with previous investigators, the Merkel cells were immunoreactive for CGRP. In the guinea-pig and rat thyroid gland CGRP immunoreactivity was localized in the C-cells. The distribution of CGRP immunoreactivity in the guinea-pig salivary glands is different from that previously reported for rat salivary glands. In the guinea-pig trigeminal ganglion, CGRP immunoreactivity was localized mainly in small-sized neurons and fibres traversing the ganglion. Double staining with substance P performed on guinea-pig trigeminal ganglion revealed four types of sensory neurons, those containing both peptides, those containing only substance P or CGRP and those lacking both peptides. Guinea-pig parotid gland, but not the submandibular or sublingual glands, contained periacinar fibres exhibiting both immunoreactivities. Substance P-positive, CGRP-positive fibres were also seen around parotid and submandibular, but not around sublingual, gland ducts. All glands received perivascular innervation showing immunoreactivities for both peptides. The present results support the idea that in the peripheral nervous system only a subpopulation of sensory neurons contains both substance P and CGRP. Consequently, colocalization of substance P and CGRP indicates a sensory nerve, while those containing either substance P or CGRP may be sensory or parasympathetic.     

Calcitonin gene-related peptide (CGRP)-immunoreactive sensory and motor nerves of the mammalian palate

The distribution of calcitonin gene-related peptide (CGRP)-immunoreactive nerve fibres in the palate of rat, cat and monkey was studied using immunocytochemistry and radioimmunoassay. CGRP-containing nerve fibres were found, in all species studied, to form a rich plexus in the subepithelial and submucous layers, around excretory ducts and blood vessels. A small number of CGRP-containing nerve fibres penetrated the epithelium of the hard and soft palate, and terminated as free endings. Some CGRP-containing nerve fibres were found in the vicinity of the mucous glands. CGRP-immunoreactive motor end plates were seen in the striated muscle (tensor veli palatini) of the soft palate. Following capsaicin treatment a small depletion in CGRP-immunoreactive nerve fibres in the rat palate epithelium was noted. In contrast, CGRP immunoreactive fibres forming rich plexuses in other layers of the palate, including motor end plates, were not affected. The extractable CGRP showed no significant depletion (normal animals [n = 10] 21.7 +/- 2.4 pmol/g compared with capsaicin-treated animals [n = 10] 17.5 +/- 1.8 pmol CGRP/g wet weight). The reduction in the number of visible immunoreactive nerves following capsaicin application tends to confirm the sensory character of the CGRP-containing nerve fibres terminating in the epithelium of the hard and soft palate. The capsaicin insensitive CGRP-immunoreactive nerve fibres may thus have a predominantly motor function.(ABSTRACT TRUNCATED AT 250 WORDS)     

Morphological changes of sensory CGRP-immunoreactive and sympathetic nerves in peripheral tissues following chronic denervation

Summary The morphological relationship between sensory and sympathetic nerves was studied in tissues of the eye and the oral cavity following chronic sympathetic or sensory denervation. Immunoreactivities for calcitonin gene-related peptide (CGRP) and tyrosine hydroxylase (TH) were used as indexes to assess the changes of the two nerve populations after denervation.Following surgical sympathectomy, a marked increase of CGRP-containing fibres was seen in all tissues studied, while TH-imunoreactive fibres were totally depleated. Conversely, after capsaicin treatment, an increase of TH-immunoreactive nerves was found in the same tissues, concomitant with a sharp decrease of CGRP-immunoreactive nerves. These changes were particularly evident in iridial stroma and around blood vessels in all tissue, where sensory and sympathetic nerves have a closely overlapping distribution pattern.The altered proportion of sensory peptide-and catecholamine-containing nerves following sympathetic and sensory denervation suggest that there is a reciprocal trophic influence between the two nerve subsets, possibly with the intervention of neurotrophic substances such as nerve growth factor. These results indicate a close interaction between sensory peptidergic and sympathetic nervous systems in peripheral organs.     

Morphological changes of sensory CGRP-immunoreactive and sympathetic nerves in peripheral tissues following chronic denervation

The morphological relationship between sensory and sympathetic nerves was studied in tissues of the eye and the oral cavity following chronic sympathetic or sensory denervation. Immunoreactivities for calcitonin gene-related peptide (CGRP) and tyrosine hydroxylase (TH) were used as indexes to assess the changes of the two nerve populations after denervation. Following surgical sympathectomy, a marked increase of CGRP-containing fibres was seen in all tissues studied, while TH-imunoreactive fibres were totally depleated. Conversely, after capsaicin treatment, an increase of TH-immunoreactive nerves was found in the same tissues, concomitant with a sharp decrease of CGRP-immunoreactive nerves. These changes were particularly evident in iridial stroma and around blood vessels in all tissue, where sensory and sympathetic nerves have a closely overlapping distribution pattern. The altered proportion of sensory peptide- and catecholamine-containing nerves following sympathetic and sensory denervation suggest that there is a reciprocal trophic influence between the two nerve subsets, possibly with the intervention of neurotrophic substances such as nerve growth factor. These results indicate a close interaction between sensory peptidergic and sympathetic nervous systems in peripheral organs.     

Morphologic diversity of the minor salivary glands of the rat: fertile ground for studies in gene function and proteomics

In this article the locations and histologic and ultrastructural features of all of the minor salivary glands of the rat are presented; similarities and differences among them are highlighted. These glands are almost as diverse morphologically as the major salivary glands of the rat. The acini of von Ebner's glands are serous; those of the anterior and posterior buccal glands and minor sublingual glands are mucous; and those of the glossopalatal, palatal, and Weber's glands are mucous with serous demilunes. The anterior buccal, minor sublingual and von Ebner's glands have striated and stratified columnar ducts, while only the minor sublingual and von Ebner's glands have intercalated ducts. The glossopalatal, palatal, posterior buccal and Weber's glands have none of these ducts; the tubulo-acini drain abruptly into short terminal ducts composed of stratified squamous epithelium. All of the mucous acini react with an antibody to a mucin (Muc19) of the rat major sublingual gland, but in some of the glands the reaction varies in intensity among the acinar cells. Ultrastructurally, the mucous secretory granules of the anterior buccal, glossopalatal, palatal and Weber's glands are biphasic, while those of the minor sublingual and posterior buccal glands are monophasic. Although there is a considerable body of literature concerning the development, innervation, physiology and proteomics of von Ebner's glands, investigation of the other minor salivary glands of the rat ranges from modest to nearly nonexistent.     

Pituitary adenylate cyclase-activating polypeptide in intrinsic and extrinsic nerves of the rat pancreas

Pituitary adenylate cyclase-activating polypeptide (PACAP) is the latest member of the vasoactive intestinal polypeptide (VIP) family of neuropeptides present in nerve fibres in many peripheral organs. Using double immunohistochemistry, with VIP as a marker for intrinsic innervation and calcitonin-gene related peptide (CGRP) as a marker for mainly extrinsic innervation, the distribution and localization of PACAP were studied in the rat pancreas. PACAP was demonstrated in nerve fibres in all compartments of the pancreas and in a subpopulation of intrapancreatic VIP-containing ganglion cells. PACAP and VIP were co-stored in intra- and interlobular nerve fibres innervating acini, blood vessels, and in nerve fibres within the islets of Langerhans. No PACAP immunoreactivity was observed in the islet cells. Another population of PACAP-immunoreactive nerve fibres co-localized with CGRP innervated ducts, blood vessels and acini. PACAP/CGRP-positive nerve fibres were also demonstrated within the islets. Neonatal capsaicin reduced the PACAP-38 concentration by approximately 50%, and accordingly a marked reduction in PACAP/CGRP-immunoreactive nerve fibres in the exocrine and endocrine pancreas was observed. Bilateral subdiaphragmatic vagotomy caused a slight but significant decrease in the PACAP-38 concentration compared with controls. In conclusion, PACAP-immunoreactive nerve fibres in the rat pancreas seem to have dual origin: extrinsic, most probably sensory fibres co-storing CGRP; and intrinsic, constituting a subpopulation of VIP-containing nerve cell bodies and fibres innervating acinar cells and islet cells. Our data provide a morphological basis for the reported effects of PACAP in the pancreas and suggest that PACAP-containing nerves in the rat pancreas may have both efferent and sensory functions.     

Morphologic diversity of the minor salivary glands of the rat: fertile ground for studies in gene function and proteomics

In this article the locations and histologic and ultrastructural features of all of the minor salivary glands of the rat are presented; similarities and differences among them are highlighted. These glands are almost as diverse morphologically as the major salivary glands of the rat. The acini of von Ebner's glands are serous; those of the anterior and posterior buccal glands and minor sublingual glands are mucous; and those of the glossopalatal, palatal, and Weber's glands are mucous with serous demilunes. The anterior buccal, minor sublingual and von Ebner's glands have striated and stratified columnar ducts, while only the minor sublingual and von Ebner's glands have intercalated ducts. The glossopalatal, palatal, posterior buccal and Weber's glands have none of these ducts; the tubulo-acini drain abruptly into short terminal ducts composed of stratified squamous epithelium. All of the mucous acini react with an antibody to a mucin (Muc19) of the rat major sublingual gland, but in some of the glands the reaction varies in intensity among the acinar cells. Ultrastructurally, the mucous secretory granules of the anterior buccal, glossopalatal, palatal and Weber's glands are biphasic, while those of the minor sublingual and posterior buccal glands are monophasic. Although there is a considerable body of literature concerning the development, innervation, physiology and proteomics of von Ebner's glands, investigation of the other minor salivary glands of the rat ranges from modest to nearly nonexistent.     

Calcitonin gene-related peptide (CGRP) in capsaicin-sensitive substance P-immunoreactive sensory neurons in animals and man: distribution and release by capsaicin

The presence of calcitonin-gene related peptide (CGRP)-like immunoreactivity (-LI) in sensory neurons was established by immunohistochemistry and radioimmunoassay (RIA) in combination with high performance liquid chromatography (HPLC). CGRP-immunoreactive (-IR) nerve fibres were present in many peripheral organs including heart, ureter, uterus and gall bladder of guinea-pig and man. The distribution of CGRP-IR nerves in the dorsal horn of the spinal cord, of positive cell bodies in thoracic spinal and nodose ganglia and nerves in peripheral organs was closely related to that of substance P-LI. Double staining experiments revealed that in most cases peripheral CGRP-IR nerve terminals also contained SP-LI. However, different localization of SP- and CGRP-IR neurons was observed in the nucleus of the solitary tract as well as in the ventral horn of the spinal cord. In the heart, CGRP-IR nerves were associated with myocardial cells (mainly atria), coronary vessels, local parasympathetic ganglia as well as with the epi- and endocardia. Three to 4-fold higher levels of native CGRP-LI were observed in the atria than in the ventricles of the heart. HPLC analysis revealed that the major peak of CGRP-LI in the heart of rat and man had the same retention times as the synthetic equivalents. Systemic capsaicin pretreatment and adult guinea-pigs caused a loss of CGRP-IR terminals in the dorsal horn of the spinal cord as well as in peripheral organs including the heart. After capsaicin treatment, the content of CGRP-IR was reduced by 70% in the heart and by 60% in the dorsal part of the spinal cord. In superfusion experiments with slices from the rat spinal cord, a release of CGRP-LI was induced by 60 mM K+ and 3 microM capsaicin in a calcium-dependent manner.     

Innervation of the bovine naso-labial glands

An investigation was carried out on the innervation of bovine naso-labial glands. Histochemical techniques were used to establish the presence and distribution of cholinergic fibres and peptidergic fibres containing VIP and Substance P were revealed by immunohistochemical methods. Parasympathetic nerve fibres were localized around the secretory units and intralobular excretory ducts. VIPergic nerve fibres, the only peptidergic fibres encountered, were arranged in a similar pattern. Adrenergic fibres were only seen in the periglandular connective tissue around blood vessels of medium size. We are, therefore, of the opinion that the secretory activity is mainly regulated by the parasympathetic nerves and that the VIP acts as a modulator.     

Vasoactive intestinal polypeptide (VIP)-like immunoreactivity in salivary glands.

VIP-like immunoreactivity was found in rat, cat and human salivary glands at a concentration of 17.0 – 29.3 pmol/g of tissue. The immunoreactivity was localised by immunocytochemistry in beaded nerve fibres which occurred in association with secretory acini, ducts, blood vessels and larger nerves. In salivary glands VIP may act as a neurotransmitter or neuromodulator involved in the regulation of blood flow and in the composition and volume of saliva.     

Tissue distribution and innervation pattern of peptide immunoreactivities in the rat pancreas.

The distribution of calcitonin gene-related peptide (CGRP), substance P/tachykinin (SP/TK), vasoactive intestinal polypeptide (VIP), neuropeptide Y (NPY) and gastrin-releasing peptide (GRP) immunreactivities (IR) in the rat pancreas was investigated using radioimmunoassay and immunohistochemistry. CGRP, NPY and VIP tissue contents are much higher than GRP and SP/TK concentrations. Peptide-containing nerves are distributed to both the exocrine and endocrine pancreas. However, differences exist in terms of density and targets of innervation for each peptidergic system. In the acini and through the stroma, fibers IR for CGRP, NPY and VIP are greater than GRP- and SP/TK-containing processes. The vasculature is supplied by a prominent NPY, CGRP and, to a lesser extent, SP/TK innervation. VIP-IR is found occasionally, and GRP-IR is never detected, in fibers associated with blood vessels. Around ducts, CGRP- and NPY-positive neurites are greater than SP/TK- greater than or equal to VIP-IR fibers, whereas GRP-containing nerves are not visualized. In the islets, the density of peptidergic nerves is: VIP-, GRP- greater than or equal to CGRP-IR greater than NPY or SP/TK. In intrapancreatic ganglia. VIP- and, to a lesser extent, NPY-IRs are found in numerous neuronal cell bodies and in nerve fibers; GRP-IR is present in numerous nerve processes and in few cell bodies; CGRP- and SP/TK-IRs are detected only in fibers wrapping around unlabeled ganglion cells. The majority of CGRP-IR fibers contain SP/TK-IR. The existence of differential patterns of peptidergic nerves suggests that peptides exert their effects on pancreatic functions via different pathways.     

Noradrenergic and peptidergic innervation of the mammary gland in the immature pig

The presence and pattern of coexistence of some biologically active substances in nerve fibres supplying the mammary gland in the immature pig were studied using immunohistochemical methods. The substances studied included: protein gene product 9.5 (PGP), tyrosine hydroxylase (TH), somatostatin (SOM), neuropeptide Y (NPY), galanin (GAL), calcitonin gene-related peptide (CGRP) and substance P (SP). The mammary gland was found to be richly supplied by PGP-immunoreactive (PGP-IR) nerve fibres that surrounded blood vessels, bundles of smooth muscle cells and lactiferous ducts. The vast majority of these nerves also displayed immunoreactivity to TH. Immunoreactivity to SOM was observed in a moderate number of nerve fibres which were associated with smooth muscles of the nipple and blood vessels. Immunoreactivity to NPY occurred in many nerve fibres associated with blood vessels and in single nerves supplying smooth muscle cells. Solitary GAL-IR axons supplied mostly blood vessels. Many CGRP-IR nerve fibres were associated with both blood vessels and smooth muscles. SP-IR nerve fibres richly supplied blood vessels only. The colocalization study revealed that SOM, NPY and GAL partly colocalized with TH in nerve fibres supplying the porcine mammary gland.     

Calcitonin gene-related peptide (CGRP) in the female rat urogenital tract

CGRP-immunoreactivity was found throughout the female rat urogenital tract by specific radioimmunoassay, and shown to be present in nerve fibres by immunocytochemistry. The highest concentrations of CGRP-like immunoreactivity were found in the urinary tract, with lower levels in regions of the genitalia. Chromatographic analysis of bladder and vaginal extracts on Sephadex G-50 columns and HPLC revealed at least three CGRP-immunoreactive peaks. The major peak emerged in the same position as synthetic rat CGRP. CGRP nerve fibres were associated mainly with blood vessels, non-vascular smooth muscle, squamous epithelium and uterine and cervical glands, and were particularly abundant in the ureter and bladder. CGRP-immunoreactivity was depleted by neonatal treatment with capsaicin and after surgical section of pelvic and/or hypogastric nerves. Immunocytochemistry demonstrated that depletion occurred predominantly in the mucosal layer of the urogenital tract. These findings indicate a sensory function for most of the CGRP-immunoreactive nerves in the rat urogenital tract.