Sensory Nerve Innervation of Epineurial Arterioles of the Sciatic Nerve Containing Calcitonin Gene–Related Peptide: Effect of Streptozotocin-Induced Diabetes

The authors have determined that epineurial arterioles of the sciatic nerve are innervated by nonadrenergic, noncholinergic nerves that contribute to the regulation of vasodilation. Using immunohistochemistry, the authors determined that nerves innervating epineurial arterioles contain the neuropeptide calcitonin gene–related peptide (CGRP). Using streptozotocin-induced diabetic rats, the authors demonstrated that CGRP content in sensory nerves innervating epineurial arterioles and vasodilation in response to exogenous CGRP was decreased. In summary, epineurial arterioles of the sciatic nerve are innervated by sensory nerves containing the neuropeptide CGRP. The diabetes-like condition induced by streptozotocin reduces the content of CGRP in these nerves and exogenous CGRPmediated vasodilation. CGRP is likely an important regulator of vascular tone and compromising its function could contribute to nerve ischemia and diabetic neuropathy.     

Comparative morphology,morphometry and distribution pattern of the trigeminal nerve branches supplying the bill tip in the ostrich (Struthio camelus) and emu (Dromaius novaehollandiae)

The subdivisions of the trigeminal nerve (N. ophthalmicus R. medialis and N. intramandibularis) innervating the upper and lower bill tip, respectively, were well developed in both the ostrich and emu and displayed extensive branching. Transmission electron microscopy revealed that both nerves displayed features typical of a mixed, peripheral nerve. Nerve fibre size in the ostrich and emu was larger than that reported in domestic poultry. There were a significantly higher number of myelinated nerve fibres in the N. ophthalmicus R. medialis in the emu by comparison with the same nerve in the ostrich, or by comparison with the N. intramandibularis of either species. As myelinated nerve fibres supply Herbst corpuscles, and these structures have been demonstrated in this region in these two species, this may indicate that the upper bill of the emu is more sensitive to vibrational stimuli than the upper bill of the ostrich or the lower bill of both species. The large size of the nerve fibres, the high nerve fibre count and the particular distribution of the nerves in the bill tip support the existence of a well‐developed sensory area in this region of the ostrich and emu.     

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.     

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.     

Neuropeptide Y modulates the action of vasodilator agents in guinea-pig epicardial coronary arteries

Recently, we have demonstrated that guinea-pig epicardial coronary arteries are supplied by numerous nerve fibres containing neuropeptide Y (NPY) immunoreactivity. However, examination of vasomotor responses revealed that NPY did not elicit a contractile response in these arteries. In contrast, acetylcholine (ACh), calcitonin gene-related peptide (CGRP), substance P and vasoactive intestinal polypeptide (VIP) all relaxed precontracted arteries. In the present study, we have used histochemical, immunohistochemical and in vitro pharmacological techniques, in order to further investigate the possible role of NPY in guinea-pig epicardial coronary arteries. A double-immunofluorescence staining technique revealed that CGRP and substance P were co-localized in nerve fibres distinct from those displaying NPY immunoreactivity. Furthermore, using a method combining immunofluorescence and histochemical techniques, we observed that putative cholinergic nerve fibres (identified by their acetylcholinesterase content) and NPY-immunoreactive nerve fibres are two different nerve populations. An in vitro pharmacological method demonstrated that NPY markedly inhibited the relaxant responses mediated by ACh, VIP, substance P and isoprenaline but had no effect on CGRP. These results suggest that NPY-containing nerves associated with guinea-pig epicardial coronary arteries may be predominantly involved in modulating the action of vasodilator agents.     

An immunocytochemical study of cutaneous innervation and the distribution of neuropeptides and protein gene product 9.5 in man and commonly employed laboratory animals.

The cutaneous nerves of rat, cat, guinea pig, pig, and man were studied by immunocytochemistry to compare the staining potency of general neural markers and to investigate the density of nerves containing peptides. Antiserum to protein gene product 9.5 (PGP 9.5) stained more nerves than antisera to neurofilaments, neuron-specific enolase (NSE), and synaptophysin or histochemistry for acetylcholinesterase (AChE). Peptidergic axons showed species variation in density of distribution and were most abundant in pig and fewest in man. However, the specific peptides in nerves innervating the various structures were consistent between species. Nerve fibers immunoreactive for calcitonin gene-related peptide (CGRP) and/or vasoactive intestinal polypeptide (VIP) predominated in all the species; those immunoreactive to tachykinins (substance P and neurokinin A [NKA]) and neuropeptide tyrosine (NPY) were less abundant. Neonatal capsaicin, at the doses employed in this study, destroyed approximately 70% of CGRP- and tachykinin-immunoreactive sensory axons; whereas 6-hydroxydopamine (6-OHDA) at the doses employed resulted in a complete loss of NPY and tyrosine hydroxylase (TH) immunoreactivity without affecting VIP, CGRP, and tachykinins. Thus, this study confirms that antiserum to PGP 9.5 is the most suitable and practical marker for the demonstration of cutaneous nerves. Species differences exist in the density of peptidergic innervation, but apparently not for specific peptides. Not all sensory axons immunoreactive for CGRP and substance P/NKA are capsaicin-sensitive. However, all sympathetic TH- and NPY-immunoreactive axons are totally responsive to 6-OHDA; but no change was seen in VIP-immunoreactive axons, suggesting some demarcation of cutaneous adrenergic and cholinergic sympathetic fibers.     

Calcitonin gene-related peptide (CGRP) immunoreactivity in the neuroendocrine Merkel cells and nerve fibres of pig and human skin

Summary The presence of calcitonin gene-related peptide (CGRP) in the skin of pig snout and human fingertip was investigated using immunohistochemical techniques. CGRP immunoreactivity was found in Merkel cells and nerve fibres of both species. In pig snout skin, Merkel cells containing CGRP were seen forming clusters at the tips of rete ridge epidermis and in the external root sheath of sinus hair follicles (vibrissae). Human Merkel cells immunostained for CGRP were found isolated or forming small groups in the basal layer of glandular epidermal ridges. In all cases, immunoreactivity was more intense on the side of the Merkel cell facing the associated nerve terminal (which was never positive for CGRP). This part of the Merkel cell has the greatest density of dense-cored granules, suggesting that CGRP must be stored in these granules. Nerve, bundles containing CGRP-immunoreactive fibres were found at dermal and hypodermal level, and blood vessels were often surrounded by CGRP nerve fibres. In pig snout skin some nerve fibres containing CGRP penetrated the epidermis and terminated as free endings, and in the human fingertip a small number of CGRP-immunoreactive nerve fibres were seen in Meissner's corpuscles.     

Calcitonin gene-related peptide (CGRP) immunoreactivity in the neuroendocrine Merkel cells and nerve fibres of pig and human skin

The presence of calcitonin gene-related peptide (CGRP) in the skin of pig snout and human fingertip was investigated using immunohistochemical techniques. CGRP immunoreactivity was found in Merkel cells and nerve fibres of both species. In pig snout skin, Merkel cells containing CGRP were seen forming clusters at the tips of rete ridge epidermis and in the external root sheath of sinus hair follicles (vibrissae). Human Merkel cells immunostained for CGRP were found isolated or forming small groups in the basal layer of glandular epidermal ridges. In all cases, immunoreactivity was more intense on the side of the Merkel cell facing the associated nerve terminal (which was never positive for CGRP). This part of the Merkel cell has the greatest density of dense-cored granules, suggesting that CGRP must be stored in these granules. Nerve bundles containing CGRP-immunoreactive fibres were found at dermal and hypodermal level, and blood vessels were often surrounded by CGRP nerve fibres. In pig snout skin some nerve fibres containing CGRP penetrated the epidermis and terminated as free endings, and in the human fingertip a small number of CGRP-immunoreactive nerve fibres were seen in Meissner's corpuscles.     

Calcitonin gene-related peptide-receptor component protein expression in the uterine cervix, lumbosacral spinal cord, and dorsal root ganglia

The neuropeptide calcitonin gene-related peptide (CGRP) may play a role in neurogenic inflammation, tissue remodeling of the uterine cervix, promoting vasodilation, parturition, and processing of sensory information in the spinal cord. CGRP-immunoreactive nerves of the cervix and spinal cord have been studied but cellular identification of the CGRP receptor has received little attention. CGRP-receptor component protein (CGRP-RCP) is a small protein associated with the CGRP receptor; thus, immunostaining for the CGRP-RCP can be used to identify sites of the CGRP receptor. We determined sites of CGRP-RCP immunoreactivity relative to the presence of CGRP-ir nerve fibers in the female rat uterine cervix, spinal cord, and dorsal root ganglia. CGRP-RCP immunoreactivity was expressed in the dorsal horn of the spinal cord, venules of the uterine cervix, and perikarya of sensory neurons in dorsal root ganglia. CGRP-immunoreactive fibers were adjacent to CGRP-RCP-immunoreactive vessels in the cervix and among CGRP-RCP-immunoreactive structures in the dorsal horn of the spinal cord. This suggests CGRP-RCP is associated with structures innervated by CGRP nerves and these interactions may be changed in tissues in response to an appropriate stimulus.     

Nerve growth factor receptor-like immunoreactivity in primary and permanent canine tooth pulps of the cat

Summary The distribution of nerve growth factor receptor (NGF receptor)-like immunoreactivity in pulps of developing primary and mature permanent cat canine teeth was examined, by use of a monoclonal antibody against NGF receptor detected by fluorescence immunohistochemistry and pre-embedding immunocytochemical light- and electron microscopy. Both primary and permanent pulps contained a vast number of NGF receptor-like immunoreactive nerves. Immunolabelling appeared to be localized both to axons and Schwann cells. In addition, many blood vessel walls in immature primary tooth pulps showed NGF receptor-like immunoreactivity, in contrast to permanent pulps where blood vessels rarely were NGF receptor-immunoreactive. Double-labelling immunofluorescence experiments revealed that in the permanent pulp a majority of the NGF receptor-positive nerves also showed calcitonin gene-related peptide (CGRP)-like immunoreactivity, and many showed substance P-like immunoreactivity. However, nerve fibers with neuropeptide Y-like immunoreactivity lacked NGF receptor-like immunoreactivity. In developing primary tooth pulps fewer NGF receptor-positive nerves were CGRP-like immunoreactive or substance P-like immunoreactive, as compared to the permanent pulp. Neuropeptide Y-like immunoreactive nerve fibers were not detected in the primary tooth pulp. The results suggest a role for nerve growth factor in both developing and mature sensory nerves of the tooth pulp.     

The distribution and colocalization of neuropeptides and catecholamines in nerves supplying the gall bladder of the toad,Bufo marinus

The indirect immunofluorescence technique was used to determine the distribution of peptide-containing axons in the gall bladder of the cane toad, Bufo marinus. In addition, the adrenergic innervation of the gall bladder was examined by use of immunoreactivity to the catecholamine-synthesizing enzyme, tyrosine hydroxylase, and glyoxylic acid-induced fluorescence. On the basis of peptide coexistence, two intrinsic populations of neurones and their projecting fibres could be distinguished substance P neurones and vasoactive intestine peptide neurones. Neither of these two types of neurones contained any other colocalized neuropeptides. Four populations of nerve fibres arising from cell bodies outside the gall bladder were identified: nerves containing colocalized galanin, somatostatin and vasoactive intestinal peptide; nerves containing colocalized calcitonin gene-related peptide and substance P; adrenergic nerves containing neuropeptide Y; and nerves containing only adrenaline.     

Regulatory peptides in the pancreas of two species of elasmobranchs and in the Brockmann bodies of four teleost species

Summary Immunohistochemistry was used to localize regulatory peptides in endocrine cells and nerve fibres in the pancreas of two species of elasmobranchs (starry ray,Raja radiata and spiny dogfish,Squalus acanthias), and in the Brockmann bodies of four teleost species (goldfish,Carassius auratus, brown troutSalmo trutta, rainbow trout,Oncorhynchus mykiss and cod,Gadus morhua). In the elasmobranchs, the classical pancreatic hormones somatostatin, glucagon and insulin were present in endocrine cells of the islets. In addition, endocrine cells were labelled with antisera to enkephalins, FMRF-amide, gastrin/cholecystokinin-(CCK)/caerulein, neurotensin, neuropeptide Y (NPY), and peptide YY (PYY). Nerve fibres were demonstrated with antisera against bombesin, galanin and vasoactive intestinal polypeptide (VIP). These nerve fibres innervated the walls of blood vessels, in the exocrine as well as the endocrine tissue. In the four teleost species immunoreactivity to somatostatin, insulin and glucagon was intense in the Brockmann bodies. Cells were labelled with antisera to enkephalin, neurotensin, FMRFamide, gastrin/CCK/ caerulein, NPY, PYY and VIP. Only a few nerve fibres were found with antisera against dopamine--hydroxylase (DBH, cod), enkephalin (met-enkephalin-Arg-Phe, cod), bombesin (cod), gastrin/CCK/caerulein (cod) and VIP. Galanin-like-immunoreactive fibres were numerous in the Brockmann bodies of all teleosts examined. Immunoreactivity to calcitonin gene-related peptide (CGRP), substance P, tyrosine hydroxylase (TH), and phenyl-N-methyl transferase (PNMT) could not be found in any of the species studied.     

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.     

Simultaneous visualization of neuropeptide and acetylcholinesterase nerve subpopulations in the perivascular plexus.

A simple method combining indirect immunofluorescence and histochemical techniques was developed in order to demonstrate the presence of both neuropeptide immunoreactivity and acetylcholinesterase activity in the same whole-mount preparation. It was found that the two methods can be combined without interfering with one another and may be viewed and photographed simultaneously. The guinea pig basilar artery was chosen as a model tissue. While vasoactive intestinal polypeptide immunoreactivity and acetylcholinesterase activity were found to occur in the same perivascular nerve fibres, tyrosine hydroxylase, neuropeptide tyrosine and calcitonin gene-related peptide immunoreactivity were present in distinct nerve subpopulations. It is possible using this double staining procedure, to analyse the interrelationship of putative cholinergic nerves with other components of the autonomic and sensory nervous system.     

Coexistence of calcitonin gene-related peptide and galanin immunoreactivity in female rat pelvic and lumbosacral dorsal root ganglia.

Coexistence of immunoreactivity for calcitonin gene-related peptide (CGRP) and galanin (GAL) was examined in varicose nerve endings in female rat pelvic paracervical ganglia (PG) and in perikarya of lumbosacral dorsal root ganglia (DRG). Varicose peptide-containing nerves were closely adjacent to somata of neurons in the PG, certain somata being virtually surrounded by immunoreactive varicosities. Some nerve endings were immunoreactive for either CGRP or GAL; in others, immunoreactivity for CGRP and GAL coexisted. Likewise, many perikarya in DRG were CGRP immunoreactive, fewer were GAL immunoreactive, and in some immunoreactivity for CGRP and GAL coexisted. The results suggest there are subpopulations of neuropeptide-containing sensory nerve endings in the PG; some contain CGRP, some contain GAL, and in some CGRP and GAL coexist. These substances contained in sensory nerve endings could have important roles in pelvic ganglionic functions.     

Peptidergic nerves in human dental pulp

Summary The peptidergic innervation of human dental pulp was studied with indirect immunofluorescence and immunoperoxidase techniques. Pulpal nerve fibres displaying immunoreactivity for cholecystokinin, calcitonin gene-related peptide, C-terminal flanking peptide of neuropeptide tyrosine, leucine-enkephalin, methionine-enkephalin, neuropeptide K, neuropeptide tyrosine, peptide with N-terminal histidine and C-terminal isoleucine, somatostatin-28, substance P and vasoactive intestinal polypeptide were observed. Immunoreactive axon varicosities were detectable within radicular and coronal nerve trunks and within the nerve plexus of Raschkow in the para-odontoblastic region. Many peptidergic nerve fibres were observed in association with blood vessels of various sizes. Substance P- and calcitonin-gene-related peptide-immunoreactive axons were visible in the odontoblastic layer. The occurrence of VIP- and PHI-immunoreactive fibres lends support to the hypothesis that human tooth may be supplied by parasympathetic nerves. The immunocytochemical results here shown provide a morphological basis to previous experimental studies concerning the possible roles of neuropeptides in nociception mechanisms, control of the blood flow and modulation of the inflammatory response in dental tissues.     

Peptidergic nerves in human dental pulp. An immunocytochemical study

The peptidergic innervation of human dental pulp was studied with indirect immunofluorescence and immunoperoxidase techniques. Pulpal nerve fibres displaying immunoreactivity for cholecystokinin, calcitonin gene-related peptide, C-terminal flanking peptide of neuropeptide tyrosine, leucine-enkephalin, methionine-enkephalin, neuropeptide K, neuropeptide tyrosine, peptide with N-terminal histidine and C-terminal isoleucine, somatostatin-28, substance P and vasoactive intestinal polypeptide were observed. Immunoreactive axon varicosities were detectable within radicular and coronal nerve trunks and within the nerve plexus of Raschkow in the para-odontoblastic region. Many peptidergic nerve fibres were observed in association with blood vessels of various sizes. Substance P- and calcitonin-gene-related peptide-immunoreactive axons were visible in the odontoblastic layer. The occurrence of VIP- and PHI-immunoreactive fibres lends support to the hypothesis that human tooth may be supplied by parasympathetic nerves. The immunocytochemical results here shown provide a morphological basis to previous experimental studies concerning the possible roles of neuropeptides in nociception mechanisms, control of the blood flow and modulation of the inflammatory response in dental tissues.     

Periviscerokinin-like immunoreactivity in the nervous system of the American cockroach

A highly specific polyclonal antiserum has been raised against periviscerokinin, the first neuropeptide isolated from the perisympathetic organs of insects (Predel et al. 1995). In this study, two different neuronal systems with periviscerokinin-like immunoreactivity were distinguished in the central nervous system of the American cockroach: (1) An intrinsic neuronal network, restricted to the head-thoracic region, was formed by intersegmental projecting neurons of the brain, suboesophageal ganglion and metathoracic ganglion. In addition, groups of local interneurons occurred in the proto- and tritocerebrum. (2) A typical neurohormonal system was stained exclusively in the abdomen; it was represented by abdominal perisympathetic organs which were supplied by three cell clusters located in each unfused abdominal ganglion. As revealed by nickel backfills, most neurons with axons entering the perisympathetic organs contained a periviscerokinin-like peptide. Immunoreactive fibres left the perisympathetic organs peripherally, innervated the hyperneural muscle and ran via the link nerves/segmental nerves to the heart and segmental vessels. All visceral muscles innervated by periviscerokinin-immunoreactive fibres were shown to be sensitive to periviscerokinin, whereas the hindgut gave no specific response to this peptide.     

Early increase in CGRP- and VIP-immunoreactive nerves in the skin of streptozotocin-induced diabetic rats.

We have previously shown depletion of nerves and neuropeptides in skin biopsies of diabetic patients, even in the absence of clinical signs and symptoms of sensory and autonomic neuropathy, but were unable to examine the changes occurring at an early stage of the disease. Therefore, the distribution and relative density of peptide-containing nerves was studied in streptozotocin-treated rats in order to assess the progression of neural changes in the initial stages of diabetes. Skin samples dissected from the lip and footpad of diabetic rats, 2, 4, 8 and 12 weeks after streptozotocin injection and age matched controls were sectioned and were immunostained with antisera to the neuropeptides substance P, calcitonin gene-related peptide (CGRP), vasoactive intestinal polypeptide (VIP) and neuropeptide Y (NPY), and to a general neural marker, protein gene product 9.5 (PGP 9.5). No change was apparent in the distribution or relative density of immunoreactive cutaneous nerve fibres 2, 4 and 8 weeks after streptozotocin treatment. By 12 weeks there was a marked increase in the number of CGRP-immunoreactive fibres present in epidermis and dermis, and of VIP-immunoreactive fibres around sweat glands and blood vessels. A parallel increase was seen in nerves displaying PGP 9.5 immunoreactivity. No differences were detected in nerves immunoreactive for either substance P in the epidermis and dermis, and NPY around blood vessels. The alterations in the peptide immunoreactivities may be similar in the initial stages of human diabetes.