Calcitonin gene-related peptide (CGRP) in the nipple of the rat mammary gland

The distribution of nerve fibres immunoreactive to calcitonin gene-related peptide (CGRP) was investigated by immunohistochemistry in nipples and mammary glands from lactating and non-lactating rats and compared to the immunoreactivity of other neuropeptides including substance P (SP), neuropeptide Y (NPY), vasoactive intestinal peptide (VIP) and somatostatin (SOM). The study revealed an extensive innervation of the mammary nipples, in which CGRP-immunoreactive (IR) nerve fibres were abundantly present in the epidermis, dermal connective tissue and intralobular connective tissue of the mammary gland parenchyma. Several of the dermal CGRP-IR fibres seemed to follow blood vessels, or formed ringlet-like structures. The latter were mostly observed in the dermal connective tissue of the nipple from the lactating rat and may have a mechanoreceptive function, e.g. for the suckling stimuli. The location of SP-IR appeared to be comparable to CGRP-IR, but in fewer fibres. Dense NPY-IR networks of nerve fibres were closely associated with the fascicles of smooth musculature in the core of the nipple base. In contrast, VIP-IR fibres were only sparsely present, and SOM-IR was not detected in the mammary nipples. The immunoreactive content of CGRP and SP was determined by radioimmunoassays. The total amount of immunoreactive CGRP was significantly higher in the nipples from the pregnant and the lactating rats when compared to SP. The maximum concentration of CGRP (65.9±4.0 pmol/g) measured in the nipples of the pregnant (day 10) rats exceeded almost ninefold the maximum concentration of SP (7.7±2.0 pmol/g). Thus, the immunoreactive content of CGRP in the nipples confirmed the immunohistochemical observations, and the present results strongly suggest that CGRP is a major neuropeptide in the rat nipple.     

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.     

Distribution of GABA-like immunoreactivity in myenteric plexus of carp,frog and chicken

Summary The distribution of GABA-like immunoreactivity was studied by means of indirect immunocytochemical methods in some lower vertebrate species (carp, frog, chicken). An immunoreactive network was revealed in the myenteric plexus of the alimentary canal of carp. GABA-positive nerve cells were attached closely to the fibres in the stomach. In other gut regions immunostained neurons were less frequent. Immunoreactive fibres often formed baskets on the surfaces of immunonegative neurons along the whole length of the alimentary canal. The number of immunopositive nerve fibres and pericellular baskets seemed to be lower in the mid- and hindgut than in the foregut region. A similar distribution of GABA-immunoreactivity was revealed in the frog myenteric plexus. The ganglionated foregut region possessed a relatively dense GABAergic innervation. This part of the gut contained immunostained nerve cells and fibres, while the mid- and hindgut possessed only a scanty fibre system. Chicken exhibited an extensive immunoreactive plexus for GABA, although the GABA-stained perikarya were restricted mainly to the duodenum. Further regions of the small intestine were poor in immunoreactive cell bodies, which suggests a segmental origin and arrangement of GABAergic innervation within the plexus. In all three species studied, GABA-positive fibres run into the circular muscle layer. The varicosity suggests their influence on the movement of the smooth muscles through modifying the transmitter release of neighbouring terminals.     

Distribution of peptide-containing neurons and endocrine cells in the rabbit gastrointestinal tract,with particular reference to the mucosa

Summary The distribution patterns of peptide-containing neurons and endocrine cells were mapped in sections of oesophagus, stomach, small intestine and large intestine of the rabbit, by use of standard immunohistochemical techniques. Whole mounts of separated layers of ileum were similarly examined. Antibodies raised against vasoactive intestinal peptide (VIP), substance P (SP), somatostatin (SOM), neuropeptide Y (NPY), enkephalins (ENK) and gastrin-releasing peptide (GRP) were used, and for each of these antisera distinct populations of immunoreactive (IR) nerve fibres were observed. Endocrine cells were labelled by the SP, SOM or NPY antisera in some regions.VIP-IR nerve fibres were common in each layer throughout the gastrointestinal tract. With the exception of the oesophagus, GRP-IR nerve fibres also occurred in each layer of the gastrointestinal tract; they formed a particularly rich network in the mucosa of the stomach and small intestine. Fewer nerve fibres containing NPY-IR or SOM-IR were seen in all areas. SOM-IR nerve fibres were very scarce in the circular and longitudinal muscle layers of each area and were absent from the gastric mucosa. The SP-IR innervation of the external musculature and ganglionated plexuses in most regions was rather extensive, whereas the mucosa was only very sparsely innervated. ENK-IR nerve fibres were extremely rare or absent from the mucosa of all areas, although immunoreactive nerve fibres were found in other layers.These studies illustrate the differences in distribution patterns of peptide-containing nerve fibres and endocrine cells along the gastrointestinal tract of the rabbit and also show that there are some marked differences in these patterns, in comparison with other mammalian species.     

Neuromedin U-immunoreactivity in the nervous system of the small intestine of the pig and its coexistence with substance P and CGRP

Summary In the small intestine of the pig, neuromedin U (NMU)-immunoreactivity was mainly confined to the nerve plexus of the inner submucosal and mucosal regions. After colchicine treatment, a high number of immunoreactive nerve cell bodies was observed in the plexus submucosus internus (Meissner), whereas only a low number was found in the plexus submucosus externus (Schabadasch). The plexus myentericus as well as the aganglionic nerve meshworks in the circular and longitudinal smooth muscle layers almost completely lacked NMU-immunoreactivity. Double-labeling experiments demonstrated the occurrence of distinct NMU-containing neuron populations in the plexus submucosus internus: (1) relatively large type-II neurons revealing immunoreactivity for NMU and calcitonin gene-related peptide (CGRP) and/or substance P (SP); (2) a group of small NMU- and SP-immunoreactive neurons; (3) a relatively low number of small neurons displaying immunoreactivity for NMU but not for SP. Based on its distributional pattern, it is concluded that NMU plays an important role in the regulation and control of mucosal functions.     

Distribution of GABA-like immunoreactivity in myenteric plexus of carp, frog and chicken

The distribution of GABA-like immunoreactivity was studied by means of indirect immunocytochemical methods in some lower vertebrate species (carp, frog, chicken). An immunoreactive network was revealed in the myenteric plexus of the alimentary canal of carp. GABA-positive nerve cells were attached closely to the fibres in the stomach. In other gut regions immunostained neurons were less frequent. Immunoreactive fibres often formed baskets on the surfaces of immunonegative neurons along the whole length of the alimentary canal. The number of immunopositive nerve fibres and pericellular baskets seemed to be lower in the mid- and hingut than in the foregut region. A similar distribution of GABA-immunoreactivity was revealed in the frog myenteric plexus. The ganglionated foregut region possessed a relatively dense GABAergic innervation. This part of the gut contained immunostained nerve cells and fibres, while the mid- and hindgut possessed only a scanty fibre system. Chicken exhibited an extensive immunoreactive plexus for GABA, although the GABA-stained perikarya were restricted mainly to the duodenum. Further regions of the small intestine were poor in immunoreactive cell bodies, which suggests a segmental origin and arrangement of GABAergic innervation within the plexus. In all three species studied, GABA-positive fibres run into the circular muscle layer. The varicosity suggests their influence on the movement of the smooth muscles through modifying the transmitter release of neighbouring terminals.     

Neurokinin A in cerebral vessels: characterization, localization and effects in vitro

Nerve fibres displaying neurokinin A (NKA)-immunoreactivity (IR) were seen in trigeminal nerve cell bodies and around cerebral blood vessels. NKA-positive fibres had the same general distribution as those displaying substance P (SP)-IR. Double or sequential immunostaining revealed coexistence of NKA- and SP-IR in a population of small nerve cell bodies in the trigeminal ganglion and in perivascular nerve fibres of brain vessels; both tachykinins were also noted to coexist with calcitonin gene-related peptide (CGRP)-IR. The presence of NKA- and SP-IR in cerebral vessels from guinea pig was verified by high-performance liquid chromatography and radioimmunochemistry. The levels NKA-IR were higher than those of SP-IR in cerebral vessels of rat, guinea pig and rabbit. In cat, pig, cow and human brain vessels, the levels of NKA- and SP-IR were equal. Major cerebral vessels at the base of the brain contained higher levels of NKA- and SP-IR than pial vessels on the cerebral convexities. Only low levels of NKA-IR and SP-IR were measured in choroid plexus and dura mater. Precontracted isolated arterial segments of middle cerebral (cat), basilar (rabbit, guinea pig and rat) and pial arteries (man) relaxed following the in vitro administration of NKA and SP. The responses occurred in the same concentration range; the IC50 value for NKA was, however, about 10 times higher than that for SP, while the maximum relaxation was equal. In basilar arteries from guinea pig, the peptides NKA, SP and CGRP all induced strong and potent relaxations. There was no evidence that one of the peptides might potentiate the relaxant effects in vitro of another. The present data suggest that NKA, SP and CGRP are costored and can be released together and cooperate in the mediation of vascular reactions in response to activation of the trigemino-cerebrovascular pathway.     

Localization of substance P, CGRP, VIP, neuropeptide Y, and somatostatin immunoreactive nerve fibers in the carotid labyrinths of some amphibian species.

Immunohistochemical localization of substance P (SP), CGRP, VIP, neuropeptide Y (NPY), and somatostatin (SOM) in the carotid labyrinth were compared in some species of amphibians using the peroxidase-antiperoxidase method. Immunoreactivity of SP, CGRP, VIP, and NPY was found in the nerve fibers distributed in the intervascular stroma of the carotid labyrinth. SP, CGRP, and VIP immunoreactive varicose fibers were densely distributed in the peripheral portion of the carotid labyrinth. Some SP-immunoreactive fibers were distributed similarly to CGRP-immunoreactive fibers. The density of NPY and SOM immunoreactive varicose fibers was low. No immunoreactivity of enkephalins was observed in the labyrinth. The intensities of these peptides were varied from species to species. No glomus cells showed immunoreactivity for any of the 7 peptides studied. These results suggest that the vascular regulatory function, which is one of the possible functions of the carotid labyrinth, is controlled by the peptidergic mechanisms in addition to regulation through intimate apposition of glomus and smooth muscle cells (g-s connection).     

A neurochemical characterisation of the golden hamster myenteric plexus

The neurochemical composition of nerve fibres and cell bodies in the myenteric plexus of the proventriculus, stomach and small and large intestines of the golden hamster was investigated by using immunohistochemical and histochemical techniques. In addition, the procedures for localising nitric-oxide-utilising neurones by histochemical (NADPH-diaphorase) and immunohistochemical (nitric oxide synthase) methods were compared. The co-localisation of vasoactive intestinal polypeptide and nitric oxide synthase in the myenteric plexus of all regions of the gut was also assessed. The results demonstrated the presence of nerve fibres and nerve cell bodies immunoreactive to protein gene product, vasoactive intestinal polypeptide, substance P, calcitonin gene-related peptide, tyrosine hydroxylase, 5-hydroxytryptamine and nitric oxide synthase in all regions of the gastrointestinal tract examined. The pattern of distribution of immunoreactive nerve fibres and nerve cell bodies containing the above markers was found to vary in different regions of the gut. Myenteric neurones and nerve fibres containing immunoreactivity to nitric oxide synthase and NADPH-diaphorase reactivity, however, were shown to have an identical distribution throughout the gut. In contrast to some studies on the guinea-pig and rat, the co-existence of vasoactive intestinal polypeptide and nitric oxide synthase was seen in only a small population of myenteric neurones.     

Distribution of substance p-like immunoreactivity in nerves of the gastrointestinal tract of the frog Rana esculenta L.

Summary Substance P-like immunoreactivity in the alimentary canal of the frogRana esculenta L. was studied by means of the indirect immunoperoxidase method. In all segments of the gastrointestinal tract, immunoreactivity was revealed in both the myenteric and the submucosa plexus. Stained nerve cells were observed in the myenteric plexus but not in the submucous plexus. The proximal part of the oesophagus and hindgut were free of immunoreactive perkarya. The stained nerve cells were of the Dogiel type I category in the foregut, and type II in the midgut. Both the musculature and gastrointestinal glands were supplied with immune-positive fibres. These results indicate that substance P may play similar roles in the frog gut, as described previously in mammals and fish.     

Immunocytochemical localization of neuropeptide Y-like immunoreactivity in adrenergic and non-adrenergic neurons of the rat gastrointestinal tract

The immunocytochemical location of neuropeptide Y (NPY)-like immunoreactivity (LI) within the neuronal structures of the rat gastrointestinal (GI) tract was investigated with the indirect immunofluorescence method. NPY immunoreactive neurons were found throughout all regions of the GI tract with the largest number in the duodenum. NPY immunoreactive perikarya were mainly located in the submucosal ganglia. NPY labeled processes were extensively seen in the submucosal and myenteric plexuses, smooth muscles, muscularis mucosa, mucosa and surrounding blood vessels. Following 6-hydroxydopamine (6-OHDA) treatment, NPY immunoreactive nerve fibers around blood vessels disappeared completely and the reactive fibers in other regions were reduced in number. NPY immunoreactive nerve cell bodies in the ganglionic plexuses, however, were not affected by 6-OHDA treatment. Serial sections of the coeliac ganglion showed that NPY-LI was present in cell bodies which also displayed tyrosine hydroxylase (TH) immunoreactivity. Our results suggest that NPY is abundantly contained in both adrenergic and non-adrenergic neurons of the gut and may play an important role in the regulation of the GI tract.     

Neuronal pathways in the guinea-pig lumbar sympathetic ganglia as revealed by immunohistochemistry

Summary Tyrosine hydroxylase (TH)- and peptide-immunoreactivity of postganglionic neurons and of nerve fibres in guinea pig lumbar paravertebral sympathetic ganglia 2–4 after transection of the communicating rami and the visceral branches, respectively, were investigated by single-and double-labelling techniques. Six subpopulations of postganglionic neurons were discriminated immunohistochemically: two cell types, which were immunoreactive to only one of the applied antisera — TH, and vasoactive intestinal polypeptide (VIP); and four cell types in which immunoreactivity was colocalized — TH/neuropeptide Y (NPY), NPY/VIP, dynorphin/α-neoendorphin and dynorphin (α-neoendorphin)/NPY. Small intensely fluorescent (SIF) cells dependent on their location exhibited differential immunobehaviour to NPY-/dynorphin-(α-neoendorphin-) and TH-antisera. Immunoreactivity to substance P (SP), calcitonin gene-related peptide (CGRP), met-enkephalin-arg-phe (MEAP) and leu-enkephalin was present in nerve fibres but not in postganglionic neurons with frequent colocalization of SP/CGRP- and MEAP/leu-enkephalin- and, sometimes leu-enkephalin/SP- and dynorphin/SP-immunoreactivity. TH-immunoreactive intraganglionic nerve fibres were numerically more increased after cutting the visceral branches, than after transection of the communicating rami. Vice versa, NPY-, VIP-, dynorphin- and α-neoendorphin-immunoreactive nerve fibres were particularly increased in number after cutting the communicating rami. Many but not all of the nerve fibres exhibited colocalization of two of these peptides. SP-, CGRP-, and enkephalin-immunoreactive nerve fibres were not visibly affected by cutting the visceral branches but virtually disappeared after lesioning the communicating rami.     

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     

Neuronal pathways in the guinea-pig lumbar sympathetic ganglia as revealed by immunohistochemistry

Tyrosine hydroxylase (TH)- and peptide-immunoreactivity of postganglionic neurons and of nerve fibres in guinea pig lumbar paravertebral sympathetic ganglia 2-4 after transection of the communicating rami and the visceral branches, respectively, were investigated by single- and double-labelling techniques. Six subpopulations of postganglionic neurons were discriminated immunohistochemically: two cell types, which were immunoreactive to only one of the applied antisera - TH, and vasoactive intestinal polypeptide (VIP); and four cell types in which immunoreactivity was colocalized - TH/neuropeptide Y (NPY), NPY/VIP, dynorphin/alpha-neoendorphin and dynorphin (alpha-neoendorphin)/NPY. Small intensely fluorescent (SIF) cells dependent on their location exhibited differential immunobehaviour to NPY-/dynorphin-(alpha-neoendorphin-) and TH-antisera. Immunoreactivity to substance P (SP), calcitonin gene-related peptide (CGRP), met-enkephalin-arg-phe (MEAP) and leu-enkephalin was present in nerve fibres but not in postganglionic neurons with frequent colocalization of SP/CGRP- and MEAP/leu-enkephalin- and, sometimes leu-enkephalin/SP- and dynorphin/SP-immunoreactivity. TH-immunoreactive intraganglionic nerve fibres were numerically more increased after cutting the visceral branches, than after transection of the communicating rami. Vice versa, NPY-, VIP-, dynorphin- and alpha-neoendorphin-immunoreactive nerve fibres were particularly increased in number after cutting the communicating rami. Many but not all of the nerve fibres exhibited colocalization of two of these peptides. SP-, CGRP-, and enkephalin-immunoreactive nerve fibres were not visibly affected by cutting the visceral branches but virtually disappeared after lesioning the communicating rami.     

Neuropeptide-containing nerve fibres in the human parotid gland: a semiquantitative analysis using an antibody against protein gene product 9.5

The occurrence and distribution of neuropeptide-containing fibres in the human parotid gland were examined by the peroxidase--antiperoxidase method with attention to the quality of fixation and the condition of patients. Many fibres immunoreactive for neuropeptide Y (NPY) and vasoactive intestinal polypeptide (VIP) and a moderate number of galanin- positive (GAL) fibres were distributed around the acini. A moderate number of NPY and VIP fibres were distributed around the intercalated ducts. The semiquantitative mean densities (_SD) of periacinar NPY, VIP and GAL fibres expressed as a percentage of the total protein gene product (PGP) 9.5 immunoreactive fibres were 75.62 _ 7.25%, 70.52 _ 9.33% and 41.76 _ 5.45%, respectively, whereas those of substance P (SP), calcitonin gene-related peptide (CGRP) and FMRF amide (FMRF) fibres were below 10%. The mean densities of NPY and VIP fibres around the intercalated ducts expressed as the percentage of PGP 9.5 fibres associated with these ducts were 52.37 _ 6.19% and 59.62 _ 7.02% respectively. Those of SP, CGRP, GAL, and FMRF fibres were below 10%. The densities of NPY, VIP, SP, CGRP, GAL and FMRF fibres around the striated and excretory ducts were also below 10%. In the vasculature, NPY fibres were the most prominent. Similarly, the mean density of perivascular NPY fibres was 93.76 _ 2.03%. No somatostatin or leucine or methionine enkephalin immunoreactivity was detected around the acini, duct system or blood vessels. These findings suggest that, in this gland, the periacinar NPY, VIP and GAL fibres may participate in regulating the synthesis of saliva and its secretion and that perivascular peptidergic fibres, especially NPY fibres, may be involved in controlling local blood flow This revised version was published online in November 2006 with corrections to the Cover Date.     

Effect of subcutaneous pancreatic tissue transplants on streptozotocin-induced diabetes in rats. III. Distribution of neuropeptides in normal and diabetic (host) pancreas.

This study examines whether there is a change in the pattern of distribution of cholecystokinin-octapeptide (CCK-8), calcitonin-gene-related peptide (CGRP), neuropeptide-Y (NPY), substance P (SP) and vasoactive intestinal polypeptide (VIP) in the pancreas of streptozotocin (STZ)-diabetic (host) rats after subcutaneous pancreatic transplantation. Varicose CCK-8-immunopositive nerve fibres were observed in the wall of blood vessels of both normal and diabetic host pancreata. The density of CCK-8-immunoreactive varicose nerve fibres appeared to have increased in host rat pancreas. CGRP was demonstrated in many nerve fibres located in the wall of blood vessels of both normal and host pancreas. CGRP, however, seemed to be better expressed in the nerves of host pancreas when compared to normal. The pancreata of both normal and diabetic (host) rats contained numerous NPY-immunopositive varicose nerve fibres located in the wall of blood vessels. SP was demonstrated in neurons located in the interlobular areas of normal tissue and in fine varicose nerve fibres of the interacinar region of the pancreas of STZ-induced diabetic rats with SPTG. In normal pancreatic tissue, VIP-immunopositive nerve fibres were observed in all areas of the pancreas. VIP-positive nerve fibres were still discernible especially in the interacinar regions of the pancreas of host rats. In conclusion, the pattern of distribution and density of NPY, SP and VIP in the pancreas of STZ-induced diabetic rats with SPTG is similar to that observed in normal pancreas, but the expression of CGRP and CCK-8 seemed to have increased as a result of transplantation and or diabetes.     

Neurohormonal peptides, serotonin, and nitric oxide synthase in the enteric nervous system and endocrine cells of the gastrointestinal tract of neotenic and thyroid hormone-treated axolotls (Ambystoma mexicanum)

Immunoreactivity against vasoactive intestinal polypeptide (VIP), neurotensin (NT), substance P (SP), calcitonin gene-related peptide (CGRP), gastrin/cholecystokinin (GAS/CCK), somatostatin (SOM), serotonin (SER), and nitric oxide synthase (NOS) was investigated in the gastrointestinal tract of the urodele Ambystoma mexicanum, the axolotl, by the use of immunohistochemical techniques. The study also compares the distribution patterns and frequencies of the neurohormones, and NOS in neotenic and thyroxine-treated (metamorphosed) individuals. GAS/CCK, SP, NT, SOM, and SER immunoreactivities occurred in endocrine mucosal cells and VIP, SP, CGRP, NTSER, SER, and NOS immunoreactivities in the enteric nervous system. The GAS/CCK-immunoreactive (-IR) cells were restricted to the upper small intestine. NT-IR and SP-IR endocrine cells were found in the entire gastrointestinal tract and were most prominent in the distal large intestine. The density of the SOM-IR cells decreased from the stomach toward the large intestine. SER-IR endocrine cells were found throughout the gastrointestinal tract, with particularly high densities in the stomach and distal large intestine. The VIP-IR enteric nerve fibers were the most prominent ones, present in all layers of the entire gastrointestinal tract, and supplied the smooth muscle and the vasculature. The SER-IR fibers exhibited similar distribution patterns but were less numerous. Very few NT-IR but many SP-IR fibers were found in the muscle and submucosal layers. The NT-IR fibers mainly supplied blood vessels, while the SP-IR fibers were also in contact with the smooth muscle. In the muscle and submucosal layers, CGRP-IR fibers were associated to the vasculature; CGRP immunoreactivity occurred also in a minority of SP-IR fibers. NOS-IR nerve fibers were in contact with submucosal arteries but were the least frequent . After metamorphosis provoked by exogenous thyroxine, the number of SOM-IR endocrine cells in the stomach mucosa was increased as well as the density of VIP-IR, SER-IR, and SP-IR nerve fibers in the gastrointestinal tract. It is proposed that the observed increases may reflect refinements of the neurohormonal system after metamorphosis.     

FMRFamide-related peptides in the gut of Locusta migratoria L.: a comprehensive map and developmental profile

The gut tissues and associated nervous system of the African migratory locust, Locusta migratoria, were found to contain FMRFamide-like immunoreactive (FLI) material throughout the five larval instars and 2 weeks into the adult stage in both males and females. FMRFamide-like immunoreactivity associated with the locust gut was described using camera lucida techniques. FMRFamide-like immunoreactivity is observed in the frontal connectives, recurrent nerve, and oesophageal nerves; projections from the ingluvial ganglion onto the anterior midgut, and from the proctodeal nerve onto the hindgut and posterior midgut; in the neuropils of the frontal ganglion, hypocerebral ganglion and ingluvial ganglia; 30 cell bodies in the frontal ganglion; multipolar sensory cells on the foregut; and endocrine-like cells in the gastric caecae and midgut. Radioimmunoassay (RIA) was used to determine the quantities of FLI material in foreguts, gastric caecae, anterior and posterior midguts, and hindgut of first-fifth instar larvae, 1-3- and 14-17-day male and female adult locusts. As expected, as the tissue size (assessed by total protein content) increases, so does the amount of FLI material in each tissue. Normalizing for tissue size reveals significant differences in FLI content among the stages for each tissue tested. Reversed phase-high pressure liquid chromatography (RP-HPLC) followed by RIA has identified four groups of FLI fractions present in the gut, and different members of these groups are present in the various gut tissues.     

Immunocytochemical demonstration of vertebrate neuropeptides in the earthworm Lumbricus terrestris (Annelida,Oligochaeta)

Summary The localisation and distribution of 10 vertebrate-derived neuropeptides in the earthworm, Lumbricus terrestris, have been determined by an indirect immunofluorescence technique. The peptides are pancreatic polypeptide (PP), peptide tyrosine tyrosine (PYY), neuropeptide Y (NPY), glucagon (C-terminal), vasoactive intestinal polypeptide (VIP), peptide histidine isoleucine (PHI), gastrinreleasing peptide (GRP), calcitonin gene-related peptide (CGRP), neurotensin (NT), and met-enkephalin. For 6 of the peptides — PYY, NPY, PHI, glucagon, GRP and CGRP — this is the first demonstration of their presence in any annelid, and NT has not previously been described in an oligochaete. Cell bodies and nerve fibres immunoreactive to the 10 peptides occur throughout the CNS. In the PNS, epidermal sensory cells displayed immunoreactivities to PP and PYY, and PP-, PYY-, NPY-, PHI- and GRP-like immunoreactivities occurred in nerve fibres supplying the main body muscles. Nerve fibres immunoreactive to PP and PYY are also associated with the innervation of the gut (pharynx, oesophageal glands, and mid and posterior regions of the intestine). No endocrine cells immunoreactive for any of the antisera tested could be identified in the gut epithelium, suggesting that dual location of peptides in the brain and gut epithelium is a phenomenon that occurred at a later stage in evolution. No immunoreactive elements were detected in any of the organs and ducts of the reproductive and excretory systems.