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Alpha-synuclein (α-syn) is a presynaptic neuronal protein and its structural alterations play an important role in the pathogenesis of neurodegenerative diseases, such as Parkinson’s disease (PD). It has been originally described in the brain and aggregated α-syn has also been found in the peripheral nerves including the enteric nervous system (ENS) of PD patients. ENS is a network of neurons and glia found in the gut wall which controls gastrointestinal function independently from the central nervous system. Moreover, two types of epithelial cells are crucial in the creation of an interface between the lumen and the ENS: they are the tuft cells and the enteroendocrine cells (EECs). In addition, the abundant enteric glial cells (EGCs) in the intestinal mucosa play a key role in controlling the intestinal epithelial barrier. Our aim was to localize and characterize the presence of α-syn in the normal human jejunal wall. Surgical specimens of proximal jejunum were collected from patients submitted to pancreaticoduodenectomy and intestinal sections underwent immunohistochemical procedure. Alpha-syn has been found both at the level of ENS and the epithelial cells. To characterize α-syn immunoreactive epithelial cells, we used markers such as choline acetyltransferase (ChAT), useful for the identification of tuft cells. Then we evaluated the co-presence of α-syn with serotonin (5-HT), expressed in EECs. Finally, we used the low-affinity nerve growth factor receptor (p75NTR), to detect peripheral EGCs. The presence of α-syn has been demonstrated in EECs, but not in the tuft cells. Additionally, p75NTR has been highlighted in EECs of the mucosal layer and co-localized with α-syn in EECs but not with ChAT-positive cells. These findings suggest that α-syn could play a possible role in synaptic transmission of the ENS and may contribute to maintain the integrity of the epithelial barrier of the small intestine through EECs.

Cholinergic systems play a role in basic cerebral functions and its dysfunction is associated with deficit in neurodegenerative disease. Mechanisms involved in human brain diseases, are often approached by using fish models, especially cyprinids, given basic similarities of the fish brain to that of mammals. In the present paper, the organization of central cholinergic systems have been described in the cyprinid Cyprinus carpio, the common carp, by using specific polyclonal antibodies against ChAT, the synthetic enzyme of acetylcholine, that is currently used as a specific marker for cholinergic neurons in all vertebrates. In this work, serial transverse sections of the brain and the spinal cord were immunostained for ChAT. Results showed that positive neurons are present in several nuclei of the forebrain, the midbrain, the hindbrain and the spinal cord. Moreover, ChAT-positive neurons were detected in the synencephalon and in the cerebellum. In addition to neuronal bodies, afferent varicose fibers were stained for ChAT in the ventral telencephalon, the preoptic area, the hypothalamus and the posterior tuberculum. No neuronal cell bodies were present in the telencephalon. The comparison of cholinergic distribution pattern in the Cyprinus carpio central nervous system has revealed similarities but also some interesting differences with other cyprinids. Our results provide additional information on the cholinergic system from a phylogenetic point of view and may add new perspectives to physiological roles of cholinergic system during evolution and the neuroanatomical basis of neurological diseases.

Alpha synuclein (α-syn) is a 140 amino acid vertebrate-specific protein, highly expressed in the human nervous system and abnormally accumulated in Parkinson's disease and other neurodegenerative disorders, known as synucleinopathies. The common occurrence of α-syn aggregates suggested a role for α-syn in these disorders, although its biological activity remains poorly understood. Given the high degree of sequence similarity between vertebrate α-syns, we investigated this proteins in the central nervous system (CNS) of the common carp, Cyprinus carpio, with the aim of comparing its anatomical and cellular distribution with that of mammalian α-syn. The distribution of α-syn was analyzed by semiquantitative western blot, immunohistochemistry, and immunofluorescence by a novel monoclonal antibody (3D5) against a fully conserved epitope between carp and human α-syn. The distribution of 3D5 immunoreactivity was also compared with that of choline acetyltransferase (ChAT), tyrosine hydroxylase (TH), and serotonin (5HT) by double immunolabelings. The results showed that a α-syn-like protein of about 17 kDa is expressed to different levels in several brain regions and in the spinal cord. Immunoreactive materials were localized in neuronal perikarya and varicose fibers but not in the nucleus. The present findings indicate that α-syn-like proteins may be expressed in a few subpopulations of catecholaminergic and serotoninergic neurons in the carp brain. However, evidence of cellular colocalization 3D5/TH or 3D5/5HT was rare. Differently, the same proteins appear to be coexpressed with ChAT by cholinergic neurons in several motor and reticular nuclei. These results sustain the functional conservation of the α-syn expression in cholinergic systems and suggest that α-syn modulates similar molecular pathways in phylogenetically distant vertebrates.

An immunohistochemical study was conducted on the ontogeny of pituitary adenylate cyclase-activating polypeptide-27 (PACAP) immunoreactive elements within the extrinsic and intrinsic nerve supply of the chicken embryo gut. The first PACAP-immunoreactivity was detected in the extrinsic nerve supply at E 4 within the pharyngeal region and the primary sympathetic chain. At E 5.5 it appeared in the vagus nerve, the spinal cord, the secondary sympathetic chain, some perivascular plexuses and the Remak ganglion. In the intrinsic nerve supply, the first PACAP-immunoreactive elements were shown at E 4.5-E 5 in the mesenchymal bud of the proventriculus/gizzard. Then they gradually appeared also cranially and caudally both in myenteric and submucous plexuses.

The ontogenesis and distribution of serotonin-, chromogranin A-, chromogranin B-, galanin-, neurotensin-, bombesin- and neuropeptide Y-immunoreactive elements were studied in the chicken oesophagus during pre- and post-hatching life. Unlike positive nerve elements, that were present in pre- and post-hatching life, positive endocrine cells were observed only during embryonic life in the oesophageal epithelium. The first endocrine cells, immunoreactive for serotonin and chromogranins, appeared on day 12, in the cervical and thoracic portions of the oesophagus. At the same age, but only in its distal portion, a few bombesin- and neurotensin-immunoreactive cells also appeared. The number of the endocrine cells progressively increased, reaching a maximum on day 15. They then decreased, with a cranio-caudal progression, until they disappeared a few days after hatching. Almost all the serotonin-immunoreactive cells but only a subpopulation of bombesin- and neurotensin-immunoreactive cells colocalized chromogranins. About half of this subpopulation also colocalized serotonin. All these cells reacted positively with Grimelius argyrophile stain. The mucosa of the crop never contained positive endocrine cells. Positive nervous elements appeared first in the wall of the terminal oesophagus and only one or two days later in the proximal oesophagus including the crop. Nervous elements immunoreactive for galanin first appeared from days 6 to 7, for neurotensin from days 7 to 8, for neuropeptide Y from 13 to 15 and for bombesin from 15 to 18. At day 15 galanin-immunoreactive ganglionic cells and fibres occupied both the myenteric and submucous plexus and galanin-positive nerve fibres could be seen throughout the oesophageal wall from the adventitia to a thin subepithelial network. Neurotensin- and neuropeptide Y-immunopositive ganglionic cells and fibres, by contrast, invariably occupied the muscular and submucous layers. Scattered bombesin-immunoreactive ganglionic cells were observed only in the myenteric plexus. The number of positive nerve elements progressively increased until some weeks after birth. Density and intensity were always much higher for galanin and neurotensin than for neuropeptide Y and bombesin.

Acetylcholine, the first neurotransmitter to be identified in the vertebrate frog, is widely distributed among the animal kingdom. The presence of a large amount of acetylcholine in the nervous system of cephalopods is well known from several biochemical and physiological studies. However, little is known about the precise distribution of cholinergic structures due to a lack of a suitable histochemical technique for detecting acetylcholine. The most reliable method to visualize the cholinergic neurons is the immunohistochemical localization of the enzyme choline acetyltransferase, the synthetic enzyme of acetylcholine. Following our previous study on the distribution patterns of cholinergic neurons in the Octopus vulgaris visual system, using a novel antibody that recognizes choline acetyltransferase of the common type (cChAT), now we extend our investigation on the octopus central brain mass. When applied on sections of octopus central ganglia, immunoreactivity for cChAT was detected in cell bodies of all central brain mass lobes with the notable exception of the subfrontal and subvertical lobes. Positive varicosed nerves fibers where observed in the neuropil of all central brain mass lobes.

Alpha-synuclein is a 140 amino acids’ protein, widely expressed in the nervous system of different vertebrates and closely related with several neurodegenerative disorders. Although its pathological involvement is reported from long time, its physiological function and its role in neurodegeneration is not yet clear. Disposing of two new monoclonal antibodies, able to detect alpha-synuclein in different compartments of the neurons, the aim of this study is to create an anatomical map of the protein's distribution in the central nervous system of C57 BL\6J mouse, the mouse strain most sensitive to 1-methyl 4-phenyl 1,2,3,6-tetrahydro pyridine neurotoxicity and widely used to apply toxic models of Parkinson disease. The two monoclonal antibodies confirm their ability in visualizing the protein in distinct compartments of the neurons, since 2E3 detects alpha-synuclein in the nerve cells’ fibers, whereas 3D5 preferentially in the neuronal nuclei. Both antibodies, instead, are able to show alpha-synuclein at the synaptic terminals. The protein is ubiquitary distributed in the brain, as well as in the spinal cord, but its sub-cellular localization differs markedly in the various regions of the central nervous system. Among alpha-synuclein immunoreactive territories, we describe a particular organization in habenular nuclei, dorsal hippocampus, olfactory bulbs, brain stem nuclei and cerebellar cortex. This preliminary immunohistochemical study, provides the first anatomical map of the alpha-synuclein distribution in the C57 BL\6J mouse CNS and suggests that alpha-synuclein is differentially localized, at sub-cellular level, in different types of neurons and that, therefore, it can plays a specific role for each neuronal subtype. Our study in healthy C57 BL/6J mice represents a starting point to analyze the variations in the overall distribution of alpha-synuclein in mouse models of Parkinson disease.

Intestinal motility disorders are an important problem in the postoperative management of patients with intestinal atresia. Intestinal motility could be initiated by luminal factors that activate intrinsic and extrinsic primary afferent nerves involved in the peristaltic reflex. Endocrine cells act as a key point, because they transfer information regarding the intestinal contents and intraluminal pressure to nerve fibers lying in close proximity to the basolateral surface of the epithelium. In chick embryo, experimental intestinal atresia is associated with disorders in the development of the enteric nervous system, related to the severity of intestinal dilation. Our aim was to investigate the distribution pattern of endocrine cells in the developing endocrine system of chick embryo small intestine with experimentally-induced atresia on day 12 and on day 16. Changes in enteroendocrine population were examined in gut specimens (excised proximal and distal to the atresia) from experimental embryos 19 days old and in control sham-operated chick embryos at the same age. Sections from proximal and distal bowel and control bowel were stained with Grimelius silver stain, a valuable histochemical method for detecting the argyrophil and argentophilic cells, and with an immunohistochemical procedure for detecting serotonin and neurotensin immunoreactive cells. In chick embryo proximal bowel, intestinal dilation differed in the various embryos. We found significantly higher enteroendocrine cell counts in proximal bowel than in distal and control bowel. The differences depended on the precociousness of surgery and the severity of dilation. Considering the major contribution of enteroendocrine cells to the peristaltic reflex, our data may help to explain the pathogenesis of motility disorders related to intestinal atresia.

The extrinsic and intrinsic respiratory nervous systems receive specific contributions from the vagal and sympathetic components. Using specific markers for vagal and sympathetic structures, we studied the distribution patterns of immunoreactivity to galanin (GAL), pituitary adenylate cyclase-activating polypeptide-27 (PACAP) and the tachykinin substance P in extrinsic and intrinsic nerve of chick embryo respiratory system, during development from the very early age to hatching. All peptides studied appeared in the intrinsic and extrinsic nervous systems early. We found substance P in both the vagal and sympathetic systems, PACAP in vagal components alone and GAL mainly in the sympathetic system. The intrinsic nervous system showed high immunoreactivity for all peptides studied. These data accord with the well known early trophic functions that peptides have on the development of nervous networks and modulatory activity on the intrinsic nervous system. The GAL again proves to be the main peptide in chick embryo sympathetic respiratory system.

Intestinal motility disorders are a common complication after surgery for neonatal intestinal atresia. Although intestinal atresia causes alterations in the enteric nervous system, especially in its inner structures (nervous fibers in the mucosa, submucous and deep muscular plexuses), how these alterations develop is unclear. The chick model is a useful research tool for investigating the ontogenesis of the enteric nervous system and the pathogenesis of congenital bowel diseases. More information is needed on the overlap between the developing enteric nervous system and intestinal atresia. Because vasoactive intestinal polypeptide and substance P are typical intestinal neuropeptides, and vasoactive intestinal polypeptide acts as a modulator in neurodevelopment and an inhibitor of smooth muscle cell proliferation, our aim in this study was to investigate the distribution of their immunoreactivity in the developing enteric nervous system of normal and experimental chick models. We studied gut specimens excised from normal chick embryos (aged 12-20 days) and experimental chick embryos (aged 15-20 days) that underwent surgical intervention on day 12 to induce intestinal atresia (atresic embryos) or simply to grasp the bowel loop (sham-operated embryos). In normal chick embryos we showed vasoactive intestinal polypeptide and substance P immunoreactivity from day 12 in the submucous and myenteric plexuses. The distribution of peptide immunoreactivity differed markedly in atresic and normal or sham-operated gut embryos. These differences especially affected the inner structures of the enteric nervous system of specimens proximal to atresia and were related to the severity of dilation. Because nerve structures in the gut wall mucosa and submucous and deep muscular plexuses play a role in motility control and stretch sensation in the intestinal wall, our findings in the chick embryo may help to explain how gut motility disorders develop after surgery for neonatal intestinal atresia.

Guanylin-like peptides regulate electrolyte/water transport through the epithelia. Moreover, these peptides possess antiproliferative activity and regulate the turnover of epithelial cells. In an earlier study we localized guanylin immunoreactivity in secretory ducts of adult rodent salivary glands. In this study we investigated the appearance and distribution pattern of this peptide during the development of rat salivary glands. Guanylin immunoreactivity appeared at the beginning of cell differentiation from solid bud, on embryonic day 17 in the submandibular and sublingual glands and after day 18 in the parotid gland. Guanylin immunoreactivity appeared first in ductal and acinar anlage: its cell distribution pattern and fate differed in these two compartments. In the duct cells guanylin immunoreactivity spread after the duct system developed, whereas in acinar cells it disappeared after cell differentiation. The guanylin immunoreactivity we detected in adult salivary duct cells accords with guanylin's role in regulating electrolyte and water transport through the various epithelia. It does so by activating guanylate cyclase-C receptor, increasing intracellular cGMP concentration, and phosphorylating the cystic fibrosis transmembrane conductance regulator (CFTR) protein by the cGMP-dependent protein kinase II. This signaling cascade couples to the ductal electrolyte/water secretion and modulates finally the electrolyte composition of the saliva. On the other hand, CFTR is also involved in mechanisms of cell growth, by regulating apoptosis, and promoting cell differentiation. The early diffuse guanylin immunoreactivity we observed in ducts and acinar anlage, before the secretory set is operative, suggests guanylin has a role in cell differentiation.

To elucidate the main ontogenetic steps of galanin immunoreactivity within the extrinsic nerve supply of the alimentary tract, we undertook an immunohistochemical study of chicken embryo specimens. Fluorescence and streptavidin-biotin-peroxidase protocols were combined, using a galanin polyclonal antiserum, on transverse serial sections obtained from chicken embryos from embryonic Day 3 (E3) to hatching, and from 9-day-old newborn chicks. Galanin-immunoreactive cells were first detected at E3.5 within the pharyngeal pouch region, the nodose ganglion, the primary sympathetic chain, primitive splanchnic branches and the caudal portion of the Remak ganglion. At E5.5 galanin-immunoreactive cells and fibers appeared in the secondary (paravertebral) sympathetic chain, splanchnic nerves, peri- and preaortic plexuses, adrenal gland anlage and visceral nerves. Galanin-immunoreactive cells also lay scattered along the vagus nerve, and in the intermediate zone of the thoracolumbar spinal cord. At E18, galanin-immunoreactive cells and fibers were found along the entire Remak ganglion and around the gastrointestinal blood vessels. In post-hatching-9-day old chicks, the para- and prevertebral ganglia, but not the intermediate zone of the spinal cord, contained galanin-immunoreactive cells. Data indicate the presence of a consistent “galaninergic” nerve system supplying the chick embryonal gut wall. Whether this system has growth or differentiating role remains to be demonstrated. Its presence and distribution pattern in the later stages clearly support its well known role as a visceral neuromodulator of gut function. Anat Rec 262:266–278, 2001. © 2001 Wiley-Liss, Inc.

The intestinal peptides guanylin and uroguanylin regulate the electrolyte/water transport in the gastrointestinal epithelium via activation of cystic fibrosis transmembrane conductance regulator (CFTR), the cystic fibrosis gene product. Because a major but incompletely understood function of the salivary glands is the CFTR-mediated secretion of an electrolyte-rich fluid, we investigated the rat and guinea pig parotid and submandibular glands for expression, cellular distribution, and subcellular localization of guanylin and uroguanylin. RT-PCR analyses with guanylin and uroguanylin-specific primers revealed that both peptides are highly expressed in the parotid and submandibular glands. At the translational level, western blotting analyses with peptide-specific guanylin and uroguanylin antibodies identified the expected 12.5-kDa immunoreactive peptides in these organs. At the cellular level, guanylin and uroguanylin were exclusively confined to epithelial cells of the intralobular and interlobular ducts. At the subcellular level, the immunoreactivities were localized by preembedding immunoelectron microscopy to small vesicles which were concentrated at the apical part of the secretory epithelial cells. The expression and cell-specific localization of guanylin and uroguanylin in the salivary glands indicate that these peptides may be specifically involved in the regulation of CFTR-mediated electrolyte/water secretion in the salivary gland ductal system.

Galanin is a brain-gut peptide that is present in the central and peripheral nervous systems. In the gut, it is contained exclusively in intrinsic and extrinsic nerve supplies, and it is involved overall in the regulation of gut motility. To obtain information about the ontogeny of galanin, we undertook an immunohistochemical study of chicken embryos. The time of first appearance and the distribution patterns of galanin were investigated with fluorescence and streptavidin-biotin-peroxidase (ABC) immunohistochemical protocols by using a galanin polyclonal antiserum. The various regions of the gut and the pancreas were obtained from chicken embryos aged from 3 days of incubation to hatching. All specimens were fixed in buffered picric acid-paraformaldehyde, frozen, and cut with a cryostat. Galanin-immunoreactive neuroblasts were first detected at 4 days in the mesenchyme of the proventriculus/gizzard primordium and within the Remak ganglion. They then extended cranially and caudally, reaching all of the other gut regions at 6.5 days. Galanin-immunoreactive nerve elements mainly occupied the sites of myenteric and submucous plexuses. From day 15, galanin-immunoreactive nerve fibers tended to invade the circular muscular layer and part of the lamina propria of the mucosa. In the pancreas, weak galanin-immunoreactive nerve elements were detected at 5.5 days. They tended to be distributed among the glandular lobules according to the organ differentiation. The widespread distribution during the earlier embryonic stages represents evidence indicating that the neuropeptide galanin may have a role as a differentiating or growth factor. From late embryonic life, its predominant presence in sympathetic nerves and in muscular layers fits with the functions demonstrated previously in adults of other vertebrates for galanin as a modulator of intestinal motility. Anat Rec 254:28–38, 1999. © 1999 Wiley-Liss, Inc.

A new amphibian peptide family has been isolated from the skin of a South American frog Phyllomedusa rhodei and named Tryptophyllins (TPH) because of their content in tryptophyl residue. Using an antiserum against one of these peptides, namely the pentapeptide Met-5-TPH-5-amide (PHE-PRO-PRO-TRP-MET-NH2), we observed the presence of a set of immunoreactive cells in rat adenohypophysis. These cells were far more numerous in pregnant than in normal male and non-pregnant female rats. Dual immunostainings demonstrated that, with some exceptions, almost all the TPH-like immunoreactive cells were gonadotrophs. At electron microscope both types of gonadotroph cells displayed immunoreactivity and the gold particles strongly labelled both types of granules. The Aa. advance the hypothesis that, besides the hormones themselves, the secretory granules might contain some TPH-like sequence.