Enteric nervous system α-synuclein immunoreactivity in idiopathic REM sleep behavior disorder.

OBJECTIVE: To investigate the expression of α-synuclein in colonic biopsies of patients with idiopathic REM sleep behavior disorder (iRBD) and address if α-synuclein immunostaining of tissue obtained via colonic biopsies holds promise as a diagnostic biomarker for prodromal Parkinson disease (PD). METHODS: Patients with iRBD, patients with PD, and healthy controls were prospectively recruited to undergo colonic biopsies for comparison of α-synuclein immunoreactivity patterns between the groups by using 2 different antibodies. RESULTS: There was no difference in colonic mucosal and submucosal immunostaining between groups using the 15G7 α-synuclein antibody, which was found in almost all participants enrolled in this study. By contrast, immunostaining for serine 129-phosphorylated α-synuclein (pSyn) in submucosal nerve fibers or ganglia was found in none of 14 controls but was observed in 4 of 17 participants with iRBD and 1 out of 19 patients with PD. CONCLUSIONS: The present findings of pSyn immunostaining of colonic biopsies in a substantial proportion of iRBD participants raise the possibility that this tissue marker may be a suitable candidate to study further as a prodromal PD marker in at-risk cohorts.

Evidence for neuronal and structural changes in submucous ganglia of patients with functional dyspepsia.

OBJECTIVES: An intact and well-functioning enteric nervous system is necessary to efficiently organize gut function. Functional gastrointestinal disorders are pathological entities in which gut function is impaired without a clearly established pathophysiology. On the basis of the relative ease with which intestinal biopsies can be obtained, and taking advantage of a recently developed optical recording technique, we evaluated whether functional neuronal defects exist in enteric nerves of patients with functional dyspepsia (FD). METHODS: The submucous plexus isolated from duodenal biopsies taken from FD patients and control subjects was used to functionally and morphologically examine nerves and ganglionic architecture (neurons and glial cells). In light of previous studies reporting eosinophil and mast cell infiltration in the gut mucosa of FD patients, we also examined whether these cells infiltrated the submucous plexus and whether this correlated with neuronal activity and specific clinical symptoms. RESULTS: We demonstrate that neuronal functioning is impaired in the submucous plexus of FD patients, as shown by decreased calcium responses to depolarization and electrical stimulation. Glial (S100) and neuronal (HuCD) markers show signs of gliosis, altered ganglionic architecture, and neuronal abnormalities in the submucous plexus of FD patients. We found that eosinophils and mast cells infiltrated the submucous layer of FD patients to a much larger extent than in controls. A significant correlation was found between the number of these cells and the calcium transient amplitudes measured in submucous ganglia. CONCLUSIONS: We provide the first direct evidence that FD is characterized by functional and structural abnormalities within the submucous ganglion plexus, which may be of future predictive and diagnostic value in the treatment of FD patients.

A study of calretinin in Hirschsprung pathology, particularly in total colonic aganglionosis.

INTRODUCTION: Calretinin, a calcium-binding protein, has been reported to be an important new marker in Hirschsprung's disease (HD). The aim is to study the diagnostic value of Calretinin in total colonic aganglionosis (TA), prematurity, and superficial biopsy when nerve hyperplasia may not be accessed by ACE activity. METHODS: Records of patients diagnosed with HD at our institution from 1985 to 2010 were studied and patients with TA identified. We examined tissue samples from those TA, partial colectomies for HD, biopsies for suspicion of HD, and rectal tissue from aborted fetuses. Immunohistochemical analysis of Calretinin was compared with ACE gold standard method in all cases. RESULTS: In the majority of the cases, the diagnosis was ascertained by ACE activity and Calretinin staining. However, in 9 cases, the diagnosis was possible with Calretinin staining but not with ACE: in 4 TA because of the absence of nerve hyperplasia, and in 5 cases because the biopsies were too superficial to examine the nerve hyperplasia. In addition, Calretinin was expressed in the gut as early as 22 gestational weeks. CONCLUSION: The use of Calretinin staining may be superior to ACE activity, particularly in the context of TA, superficial biopsies, and prematurity, allowing earlier diagnosis.

Neurochemical characterization of zinc transporter 3-like immunoreactive (ZnT3(+)) neurons in the intramural ganglia of the porcine duodenum.

The SLC30 family of divalent cation transporters is thought to be involved in the transport of zinc in a variety of cellular pathways. Zinc transporter 3 (ZnT3) is involved in the transport of zinc into synaptic vesicles or intracellular organelles. As the presence of ZnT3 immunoreactive neurons has recently been reported in both the central and peripheral nervous systems of the rat, the present study was aimed at disclosing the presence of a zinc-enriched neuron enteric population in the porcine duodenum to establish a preliminary insight into their neurochemical coding. Double- and triple-immunofluorescence labeling of the porcine duodenum for ZnT3 with the pan-neuronal marker (PGP 9.5), substance P, somatostatin, vasoactive intestinal peptide (VIP), nitric oxide synthase (NOS), leu-enkephalin, vesicular acetylcholine transporter (VAChT), neuropeptide Y, galanin (GAL), and calcitonin gene-related peptide were performed. Immunohistochemistry revealed that approximately 35, 43, and 48 % of all PGP9.5-postive neurons in the myenteric (MP), outer submucous (OSP), and inner submucous (ISP) plexuses, respectively, of the porcine duodenum were simultaneously ZnT3(+). In the present study, ZnT3(+) neurons coexpressed a broad spectrum of active substances, but co-localization patterns unique to the plexus were studied. In the ISP, all ZnT3(+) neurons were VAChT positive, and the largest populations among these cells formed ZnT3(+)/VAChT(+)/GAL(+) and ZnT3(+)/VAChT(+)/VIP(+) cells. In the OSP and MP, the numbers of ZnT3(+)/VAChT(+) neurons were two times smaller, and substantial subpopulations of ZnT3(+) neurons in both these plexuses formed ZnT3(+)/NOS(+) cells. The large population of ZnT3(+) neurons in the porcine duodenum and a broad spectrum of active substances which co-localize with this peptide suggest that ZnT3 takes part in the regulation of various processes in the gut both in normal physiology and during pathological processes.

Changes in pituitary adenylate cyclase-activating Peptide 27-like immunoreactive nervous structures in the porcine descending colon during selected pathological processes.

This study reports on changes in the pituitary adenylate cyclase-activating peptide 27-like immunoreactive (PACAP-27-LI) nerve structures of the enteric nervous system (ENS) in the porcine descending colon, caused by chemically induced inflammation, nerve injury, and proliferative enteropathy (PE), which is a "natural" inflammation of the porcine digestive tract. The distribution pattern of PACAP-27-LI structures was studied using the immunofluorescence technique in the circular muscle layer, enteric plexuses (i.e., myenteric plexus (MP), outer submucous plexus (OSP), and inner submucous plexus (ISP)), and in the mucosal layer. Under physiological conditions, PACAP-27-LI perikarya have been shown to constitute 4.04 ± 0.66, 6.66 ± 0.77, and 11.19 ± 0.74 % in the MP, OSP, and ISP, respectively. Changes in PACAP-27 immunoreactivity depended on the pathological factor studied. The numbers of the PACAP-27-LI perikarya amounted to 12.26 ± 1.43, 12.28 ± 0.79, and 21.13 ± 1.19 % in chemically induced colitis, 17.83 ± 0.88, 9.03 ± 1.05, and 20.72 ± 1.35 % during PE and 10.65 ± 0.82, 6.88 ± 1.04, and 14.04 ± 1.09 % after axotomy in MP, OSP, and ISP, respectively. All of the studied processes generally resulted in an increase in the number of PACAP-27-LI nerve fibers in the circular muscle and mucosal layers. The obtained results suggest that PACAP-27-LI nerve structures of ENS may participate in various pathological states within the porcine descending colon, and their functions probably depend on the type of pathological factor.

Distribution of nestin protein: immunohistochemical study in enteric plexus of rat duodenum.

The intermediate nestin filament is expressed in neural stem cells, neuroectodermal tumors and various adult tissues under situations that reproduce developmental phases, e.g., physiological renewal of certain cell types, tissue regeneration, and healing or revascularization. In the human gastrointestinal tract, nestin has been reported in glial cells and interstitial cells of Cajal. We examined by immunohistochemistry the appearance and distribution of nestin protein in enteric ganglia of rat duodenum. Through the myenteric and submucosal plexuses, a high number of nestin-positive cells were visualized in this specie. The nestin-positive cells were smaller and more numerous than enteric neurons. They were present both within and around ganglia. The results of this study suggest that the rat enteric glial cells (EGCs) are rich in nestin, a protein usually associated with dividing or migrating cells and the dynamic reorganization of nestin filaments during the cell cycle. EGCs could function as enteric stem cells.

Neurochemical coding of the enteric nervous system in chagasic patients with megacolon.

Neuronal destruction has been considered the hallmark of pathogenic mechanisms in chagasic megacolon. Characterization of neuropeptides in the enteric nervous system from chagasic patients with megacolon could elucidate some aspects of the development of this syndrome. In the present work we demonstrate the changes in expression of neuropeptides and neurochemical markers present in neuronal plexuses from the colons of chagasic patients with megacolon. Sections of frozen tissue samples were immunohistochemically labeled for anticalretinin, cChaT, substance P, VIP, NOS, and NPY. Immunoreactivity was observed using a confocal microscope. Our results demonstrate that in chagasic patients with megacolon, inhibitory motor neurons (VIP and NOS immunoreactive) are preferentially destroyed by Trypanosoma cruzi and/or the inflammatory process. These results suggest a selective destruction of enteric neurons in the colon of chagasic patients with megacolon, pointing to an important discovery in the mechanism of pathogenesis of Chagas' disease.

Chemical coding of submucosal type V neurons in porcine ileum.

In this study, we attempted to determine the proportion of type V neurons relative to the putative whole neuron population in the two submucosal plexuses of pigs identified by their neurofilament immunoreactivity. The total neuron number was estimated in cuprolinic blue (CB)/anti-Hu protein (HU) costained wholemounts as the sum of the number of CB+/HU+, CB+/HU- and CB-/HU+ neurons. In the external submucosal plexus (ESP), HU labelled 98.6% and CB 97.3% of neurons. In the internal submucosal plexus, HU labelled 98.3%, whereas CB only marked 92.5% of neurons. Furthermore, we investigated the chemical coding of submucosal type V neurons and searched for submucosal, non-type V neurons displaying the same chemical coding as the myenteric type V neurons described earlier, i.e. the colocalization of calcitonin gene-related peptide (CGRP) and somatostatin (SOM). In order to facilitate immunohistochemical detection of neuroactive peptides, ileal segments were pretreated with colchicine prior to fixation. Type V neurons in the ESP occurred either as single cells displaying one or few prominent dendrite(s) or within aggregates displaying a dendritic tangle. In this plexus, type V neurons amounted to between 0.9 and 1.6% of all CB-stained neurons. ESP type V neurons displayed immunoreactivities for choline acetyl transferase (95.8%) and leucine-enkephalin (73.9%). All type V neurons were negative for neuronal nitric oxide synthase. Fifty-eight percent of ESP CGRP/SOM co-immunoreactive neurons displayed type V morphology, whereas 42% were non-type V neurons. Thus, the chemical coding of ESP type V neurons is in principal similar to that of the myenteric type V neurons described earlier. In the internal submucosal plexus, we found no type V neurons. In this plexus, 0.2% of all neurons counterstained with HU displayed CGRP/SOM coreactivity. As had been observed earlier concerning the myenteric type V neurons, ESP type V neurons were also closely apposed by conspicuous accumulations of boutons reactive for the same markers as the neurons themselves. Although we cannot exclude that axons of CGRP/SOM-reactive enteric, non-type V or extrinsic neurons end synaptically on type V neurons, we suggest that the main synaptic input to type V neurons originates from other type V neurons. This presents an argument for an interneuronal role of type V neurons.

Identification and localization of estrogen receptor alpha- and beta-positive cells in adult male and female mouse intestine at various estrogen levels.

Although estrogen is implicated in the regulation of mammalian intestinal function, the presence and the distribution of estrogen receptor (ER)-positive cells in the intestine are still controversial. The present study was designed to localize ERalpha- and ERbeta-expressing cells in female and male mouse intestines immunohistochemically under various estrogen conditions, especially in female mice, ovariectomized as well at various phases of the estrous cycle. Western blot analysis detected both ERalpha (66-kDa band) and ERbeta (56-kDa band). Immunohistochemical staining of paraffin-embedded sections after antigen-retrieval treatment with autoclaving revealed staining for ERalpha in submucosal interstitial cells, and double staining identified these cells as a subtype of intestinal macrophages. The number of these cells varied according to the estrous cycle phase. Administration of 17beta-estradiol to ovariectomized mice resulted in a significant increase in the number of ERalpha-positive macrophages. On the other hand, the nuclei of nerve cells in Auerbach and Meissner plexuses were positive for both ERalpha and ERbeta, but the number of positive nerve cells was not affected by estrogen. Our results indicate that estrogen and estrogenic compounds may exert their actions on the intestine in two ways; one is through interstitial macrophages and the other is through intestinal neurons.

Topography and neurochemistry of the enteric ganglia in the proventriculus of the duck (Anas platyrhynchos).

The topographical distribution of the enteric ganglia has been investigated in the proventriculus of the duck using protein gene product 9.5 (PGP 9.5) immunohistochemistry. Myenteric ganglia were usually located between the outer longitudinal and the inner circular muscle layer. Submucous ganglia were sparsely distributed and seemed to be substituted by ganglia located in the tunica mucosa. The neurochemical profile of proventricular ganglion cells was also investigated using nicotinamide adenine dinucleotide phosphate reduced-diaphorase (NADPH-d)-histochemistry and pituitary adenylate cyclase activating peptide (PACAP)/galanin (Gal) double-labelling immunohistochemistry. The majority of mucosal ganglion cells were shown to contain the NADPH-d enzyme and both the investigated peptides. These findings provide evidence for the presence of a mucosal ganglionated plexus in the glandular stomach of birds. Moreover, the neurochemical characteristics of this plexus suggest that it plays an important role in regulating several mucosal functions and, in particular, the production and the composition of the gastric juice.

Expression of Tie-2 in human peripheral and autonomic nervous system.

Tie-2, a tyrosine kinase receptor, is essential for vascular integrity by regulating cellular adhesion between pericytes and endothelial cells. The aim of this study was to identify sites of expression of Tie-2 other than the vasculature. Tie-2 expression was first detected in human colon by Western blotting and reverse-transcription-polymerase chain reaction (RT-PCR) in tissue extracts. The presence of the Tie-2 mRNA and protein was detected by immunohistochemistry and in situ hybridization in cells of the colon myenteric and submucosal plexus, in both neuronal and Schwann cells. Tie-2 protein was also found in the nervous system of the female urogenital tract. In the human sciatic nerve and schwannoma, RT-PCR, Western blotting and immunohistochemistry analysis further confirmed the presence of Tie-2 mRNA and protein in non-autonomic peripheral nervous tissue. In conclusion, using several approaches and tissues we have demonstrated the presence of Tie-2 in human peripheral and autonomic nervous tissue, suggesting a role for Tie-2 in neural tissue. Thus, attempts to disrupt the tumour vessels by manipulation of the Tie-2 system in tumours may result in side-effects in peripheral nerves.

Morphology and distribution of nitric oxide synthase-, neurokinin-1 receptor-, calretinin-, calbindin-, and neurofilament-M-immunoreactive neurons in the myenteric and submucosal plexuses of the rat small intestine.

Characterization of the enteric neurons is vital for understanding their physiological role. We have used single and dual label fluorescence and peroxidase-based immunohistochemistry in myenteric and submucosal whole mounts from the rat small intestine to evaluate the morphology and distribution of enteric neurons immunoreactive for the following phenotypic antigens: neuronal nitric oxide synthase (NOS), neurokinin-1 receptor (NK-1R), calretinin (Calr), calbindin (Cal), and neurofilament-M (NF-M). NOS-immunoreactive neurons had Dogiel type I morphology, were abundant in the myenteric plexus compared to the submucosal plexus, and never coexpressed NK-1R immunoreactivity. NK-1R- and Calr-immunoreactive neurons had Dogiel type II morphology and were distributed comparably in both plexuses. NK-1R and Calr-immunoreactivity were coexpressed in many of the same neurons. Calbindin-immunoreactive neurons exhibited four distinct morphologies: small and large Dogiel type II neurons, Dogiel type I neurons, and small elongated neurons. These neurons were significantly fewer in number in the myenteric plexus compared to the submucosal plexus. Neurofilament-M-immunoreactive neurons had three morphologies, Dogiel type II neurons, small Dogiel type II neurons, and a less common subpopulation of small, elongated, multipolar neurons. These neurons were also fewer in number in the myenteric plexus compared to the submucosal plexus. The distribution of these phenotypic markers may assist future work that elucidates the functional activities of these enteric neurons such as control of intestinal motility and adaptation to the entry of gastric contents.

An immunohistochemical study of the organization of ganglia and nerve fibres in the mucosa of the porcine intestine.

In order to elucidate the organization of the enteric nervous system in the mucous plexus, wholemounts from six intestinal regions in six pigs were studied by vasoactive intestinal peptide, substance P, nitric oxide synthase and neurofilament proteins immunohistochemistry. The mucous plexus of both large and small intestine contained ganglia and isolated neurons. They were many and comparably larger in the caecum and colon, few in the ileum, and fewer and smaller in the jejunum. The mucous plexus was subdivided into the lamina muscularis mucosae and lamina proprial subplexuses, and based on location the latter was subdivided further in order to clarify their variations with respect to the amount, sizes and shapes of ganglia and neurons, sizes and orientation of nerve strands and immunoreactivities. Ganglia were situated at different topographical levels in the lamina muscularis mucosae subplexus, outer proprial and interglandular proprial meshworks in the lamina proprial subplexus with the majority of ganglia occurring in the outer proprial meshwork. The mucous plexus in the intestine of the pig is thus a ganglionated plexus showing marked segmental variation in the amount of intramucosal ganglia and isolated nerve cells. These new observations, calls for a re-examination of the mucous plexus to elucidate the regulatory mechanisms of importance in mucosal functions and consideration of the mucous plexus in the intestine of the pig to be one of the major ganglionated plexuses.

Increased acid-sensing ion channel ASIC-3 in inflamed human intestine.

OBJECTIVES: Acid-sensing ion channels (ASICs) are expressed by rat sensory neurons and may mediate pain associated with tissue acidosis after inflammation or injury. Our aim was to examine the molecular forms and localization of ASICs in human intestine and dorsal root ganglia using immunochemical techniques, and to measure the effects of inflammation and injury. DESIGN AND METHODS: Inflamed Crohn's disease intestine and injured human dorsal root ganglia, with appropriate controls, were studied by Western blotting and immunohistochemistry, using specific affinity-purified ASIC antibodies. RESULTS: In the Western blot, there was a significant three-fold increase in the mean relative optical density of the ASIC-3 55-kDa band (but not ASIC-1 or ASIC-2) in full-thickness inflamed intestine, as well as in separated muscle and mucosal layers. There was a corresponding trend for an increased immunoreactive density and increased number of ASIC-3-positive neurons in the myenteric and sub-mucous plexus of inflamed intestine. In dorsal root ganglia, immunoreactivity for all ASICs was restricted to a sub-population (about 50%) of small-diameter (nociceptor) sensory neurons, and was generally less intense after injury. CONCLUSIONS: Increased ASIC-3 in inflamed intestine suggests a role in pain or dysmotility, for which ASICs represent new therapeutic targets.

Calbindin immunoreactivity of enteric neurons in the guinea-pig ileum.

Previous studies have identified Dogiel type II neurons with cell bodies in the myenteric plexus of guinea-pig ileum to be intrinsic primary afferent neurons. These neurons also have distinctive electrophysiological characteristics (they are AH neurons) and 82-84% are immunoreactive for calbindin. They are the only calbindin-immunoreactive neurons in the plexus. Neurons with analogous shape and electrophysiology are found in submucosal ganglia, but, with antibodies used in previous studies, they lack calbindin immunoreactivity. An antiserum that is more effective in revealing calbindin in the guinea-pig enteric nervous system has been reported recently. In the present work, we found that this antiserum reveals the same population that was previously identified in myenteric ganglia, and does not reveal any further population of myenteric nerve cells. In submucosal ganglia, 9-10% of nerve cells were calbindin immunoreactive with this antiserum. The submucosal neurons with calbindin immunoreactivity were also immunoreactive for choline acetyltransferase, but not for neuropeptide Y (NPY) or vasoactive intestinal peptide (VIP). Small calbindin-immunoreactive neurons (average profile 130 microm2) were calretinin immunoreactive, whereas the large calbindin-immunoreactive neurons (average profile 330 microm2) had tachykinin (substance P) immunoreactivity. Calbindin immunoreactivity was seen in about 50% of the calretinin neurons and 40% of the tachykinin-immunoreactive submucosal neurons. It is concluded that, in the guinea-pig ileum, only one class of myenteric neuron, the AH/Dogiel type II neuron, is calbindin immunoreactive, but, in the submucosal ganglia, calbindin immunoreactivity occurs in cholinergic, calretinin-immunoreactive, secretomotor/vasodilator neurons and AH/Dogiel type II neurons.

Evidence supporting presence of two pacemakers in rat colon.

Intracellular microelectrodes and organ bath techniques were used to study spontaneous cyclic electrical and mechanical activity in the rat colon. Electron microscopy and immunohistochemical studies showed two major populations of interstitial cells of Cajal (ICC): one associated with Auerbach's plexus (ICC-AP) and one with the submuscular plexus (ICC-SMP). The ICC-SMP network partly adhered to the submucosa when removed and was generally strongly damaged after separation of musculature and submucosa. Similarly, longitudinal muscle removal severely damaged AP. Two electrical and mechanical activity patterns were recorded: pattern A, low-frequency (0.5--1.5 cycles/min), high-amplitude oscillations; and pattern B, high-frequency (13--15 cycles/min), low-amplitude oscillations. Pattern A was recorded in preparations with intact AP but absent in those without intact AP. Pattern B was recorded in preparations with intact SMP but was absent in those lacking SMP. With full-thickness strips, the superimposed patterns A and B were recorded in circular muscle. When longitudinal muscle mechanical activity was recorded, only pattern A was present. We conclude that two pacemakers regulate rat colonic cyclic activity: the ICC-SMP network (responsible for cyclic slow waves and small-amplitude contractions) and the ICC-AP network (which may drive the cyclic depolarizations responsible for high-amplitude contractions). This is the first report showing consistent slow wave activity in the rodent colon.

Development of the submucous plexus in the large intestine of the mouse.

In the small intestine of both embryonic birds and mammals, neuron precursors aggregrate first at the site of the myenteric plexus, and the submucous plexus develops later. However, in the large intestine of birds, the submucosal region is colonised by neural-crest-derived cells before the myenteric region (Burns and Le Douarin, Development 125:4335-4347, 1998). Using antisera that recognize undifferentiated neural-crest-derived cells (p75NTR) and differentiated neurons (PGP9.5), we examined the colonisation of the murine large intestine by neural-crest-derived cells and the development of the myenteric and submucosal plexuses. At E12.5, when the neural crest cells were migrating through and colonising the hindgut, the hindgut mesenchyme was largely undifferentiated, and a circular muscle layer could not be discerned. Neural-crest-derived cells migrated through, and settled in, the outer half of the mesenchyme. By E14.5, neural-crest-derived cells had colonised the entire hindgut; at this stage the circular muscle layer had started to differentiate. From E14.5 to E16.5, p75NTR- and PGP9.5-positive cells were observed on the serosal side of the circular muscle, in the myenteric region, but not in the submucosal region. Scattered, single neurons were first observed in the submucosal region around E18.5, and groups of neurons forming ganglia were not observed until after birth. The development of the enteric plexuses in the murine large intestine therefore differs from that in the avian large intestine.

Tissue culture of the enteric nervous system from equine ileum.

Ileal samples were harvested fresh from euthanized adult horses. The tissues were microdissected to prepare wholemount preparations for immunohistochemistry and for either explant or dissociated culture systems of the enteric nervous system. Explant culture systems were established using whole-mounts of either the submucous plexus or the muscularis externa (including the myenteric plexus). Dissociated cell cultures could only be obtained from the submucous plexus. Culture systems were maintained for up to 5 days. Immunoreactivity for a neuronal marker (Pan-N) and for glial cell markers (GFAP and S100) indicated the presence of both neurons and enteric glia in the tissue culture preparations. This is the first report of equine enteric neurons being grown in tissue culture Further refinements to the techniques will be required before this in vitro model can be used for quantitative analysis.

The nervous system of the chicken proventriculus: an immunocytochemical and ultrastructural study.

The proventriculus constitutes the glandular region of the chicken stomach. This organ is innervated by two parasympathetic networks, the myenteric and submucous plexus, and here we present a systematic study of this system by immunohistochemistry and electron microscopy. All the neurons and fibres were positive for the neural markers, protein gene product 9.5 and the amidating enzymes. Immunoreactivities for the constitutive neuronal isoform of the enzyme nitric oxide synthase and the vasoactive intestinal peptide were present in neuronal bodies suggesting an intrinsic origin for the similarly immunoreactive fibres found in the proventriculus. On the other hand, immunoreactivity to gastric inhibitory peptide was only found in varicose fibres making contact with the blood vessels and the glandular epithelium, but never in the neuronal somas, suggesting that this substance may be provided by an extrinsic nervous system whose neuronal bodies are located elsewhere. Electron microscopy revealed frequent neuromuscular and neuroepithelial connections in the muscle layers, the wall of the blood vessels and the epithelium. In addition, synapsis-like structures were identified in the proximity of cells belonging to the diffuse endocrine system, providing a new example of neuroendocrine contacts. No positivity was found for antibodies against other neural substances including somatostatin, peptide histidine-isoleucine, peptide tyrosine-tyrosine, neuropeptide tyrosine, bombesin, met-enkephalin, serotonin, substance P, galanin, calcitonin gene-related peptide and S-100 protein.

Expression of the GDNF receptors ret and GFRalpha1 in the developing avian enteric nervous system.

The formation of the enteric nervous system (ENS) from neural crest-derived cell precursors requires the growth factor glial cell line-derived neurotrophic factor (GDNF) and the receptors Ret and GDNF family receptor alpha 1 (GFRalpha1). We investigated the location(s), the timing, and the extent to which these GDNF receptors appear in the population of crest-derived precursors that form the avian ENS using immunohistochemistry and in situ hybridization. Sections and whole mounts of embryonic chick gastrointestinal tract were costained with antibodies to the receptors and to HNK-1, a marker for crest-derived cells. Neural crest-derived precursors migrate through the primitive esophagus to colonize the gizzard where an extensive cellular network forms. Ret-immunoreactivity (ir) was found in a network of cells in the gizzard at embryonic day (E)3.5. As development proceeded, Ret-immunoreactive cells appeared at progressively more caudal positions and were present in the colon at E7.5. Costaining with Ret and HNK-1 was performed to determine the number of Ret-immunoreactive cells in the crest-derived population. Ret appeared in some HNK-1 cells in the esophagus and gizzard at embryonic day (E)3.5. During development, the number of crest cells with Ret increased in the ganglia of the gizzard and small intestine. GFRalpha1-ir was also found in HNK-1 cells in the esophagus at E3.5 but did not appear in the gizzard until E4.5. Surprisingly, the colonizing vanguard of crest-derived cells lacked both Ret- and GFRalpha-ir. Between E4.5 and E6.5, the fraction of HNK-1-positive cells expressing GFRalpha1 increased considerably in the foregut. Ret and GFRalpha1 were coexpressed in many cells at E6.5, and the number of such cells increased as development progressed. In the adult, GFRalpha1 and Ret were found in the neuropil of enteric ganglia. We conclude that the population of cells expressing the receptors increases during development and persists in the adult, findings that support a neurotrophic role for GDNF in the formation and maintenance of the avian ENS.