The projections of 5-hydroxytryptamine-accumulating neurones in the myenteric plexus of the small intestine of the guinea-pig

Retrograde tracing, combined with immunohistochemistry, was used to study the projections of 5-hydroxytryptamine (5-HT)-accumulating neurones within the ileum of the guinea-pig, with confocal microscopy being used to characterise further their morphology. Two classes of neurones in the myenteric plexus, capable of taking up 5-HT or analogues, were distinguished. One class had Dogiel type I morphology with lamellar dendrites, was located on the edge or in the middle of ganglia and lacked immunoreactivity for somatostatin (SOM). The other class had smooth ovoid cell bodies with multiple filamentous dendrites and a single axon and represented a subset of the SOM-immunoreactive interneurones in the myenteric plexus. Varicosities immunoreactive for 5-HT alone, 5-HT/SOM or SOM alone were present in the myenteric ganglia. Both classes of 5-HT-accumulating neurones had long aboral projections within the myenteric plexus (up to 100 mm long) and to the submucous plexus and probably function as descending interneurones.     

Shapes and projections of tertiary plexus neurons of the guinea-pig small intestine

The axons of neurons that innervate the longitudinal muscle of the small intestine in small mammals such as rabbit, rat, guinea pig and mouse form a network, the tertiary plexus, against the inner surface of the muscle. In general, because of their substantial overlap, it has not been possible to follow the ramifications of individual axons in the tertiary plexus. In the present work, the longitudinal muscle motor neurons were filled with marker dyes through an intracellular microelectrode, and their morphologies and projections were examined in whole-mount preparations of longitudinal muscle and myenteric plexus. Most neurons that were examined were in the small intestine (ileum and duodenum), but a few were examined in the distal colon. Neurons in all regions had similar morphologies and projections. The cell bodies were amongst the smallest in myenteric ganglia, with major and minor axes of 14 microns and 25 microns (mean, n = 40) in the plane of the myenteric plexus. Each neuron had a single axon that branched extensively in the tertiary plexus, most had multiple lamellar dendrites and a few had filamentous dendrites or a mixture of filamentous and lamellar dendrites. The mean area of muscle covered by an axon and its branches extended 1.6 mm orally to anally and 1.7 mm circumferentially. The area covered was 2.8 +/- 1.9 mm2 (mean +/- SD, n = 23). From the density of occurrence of cell bodies, it can be calculated that each point in the longitudinal muscle is innervated by the processes of about 100 motor neurons and is influenced by electrotonic conduction of signals through the muscle by about 300 motor neurons.     

Calbindin-immunopositive cells are cholinergic interneurons in the myenteric plexus of rabbit ileum

The 28-kDa calcium-binding protein (calbindin) is a widely studied neuronal marker in the enteric nervous system of numerous species. Calbindin has previously been detected in myenteric neurons of rabbit ileum in which 3% of all myenteric neurons are calbindin-immunopositive. We have studied the detailed morphology and chemical coding of calbindin-immunopositive neurons in this segment of the gut. We have found calbindin immunoreactivity in both strongly and weakly stained neurons. Of these, the strongly immunoreactive neurons belong to the Dogiel type I category. These neurons project only to other ganglia and primary strands of the plexus and their processes never run to the muscle or mucosal layers. The neurons within this group are 29.5±6.6 m in length and 14.7±3.8 m in width. The second smaller group of immunoreactive cells (27%) label faintly and have different morphological properties. They are characterized by their round medium-sized cell bodies (long axis: 24.4±5.2 m; short axis: 15.5±2.9 m) and do not exhibit immunoreactivity either in their dendrites or in their axonal processes. Double-label studies show that all calbindin-immunopositive neurons lack immunoreactivity for nitric oxide synthase, vasoactive intestinal peptide and substance P but all are immunoreactive for the synthesizing enzyme of acetylcholine, choline acetyltransferase. Thus, populations of neurons containing calbindin are cholinergic interneurons in the myenteric plexus of rabbit ileum.This study was supported by grant OTKA T 34160     

Projections and chemistry of Dogiel type II neurons in the mouse colon

The physiological properties, shapes, projections and neurochemistries of Dogiel type II neurons have been thoroughly investigated in the guinea-pig intestine in which these neurons have been identified as intrinsic primary afferent neurons. Dogiel type II neurons in the myenteric ganglia of mice have similar physiological properties to those in guinea-pigs but whether other features of the neurons are similar is unknown. We have used intracellular dye-filling, retrograde tracing, immunohistochemistry and nerve lesions to determine salient features of Dogiel type II neurons of the mouse colon. Dye-filling showed that the neurons provide profuse terminal networks in the myenteric ganglia and also have axons that project towards the mucosa. Retrograde tracing and lesion studies showed that these axons provide direct innervation to the mucosa. High proportions of the neurons had immunoreactivity for calretinin, calbindin, choline acetyltransferase, the purine P2X₂ receptor and calcitonin gene-related peptide (CGRP). CGRP was the most selective marker of the neurons. Following surgery to remove an area of myenteric plexus, the CGRP-immunoreactive nerve fibres in the mucosa degenerated. Thus, Dogiel type II neurons in mice have similar shapes and projections but some differences in chemistry from those in guinea-pigs. The close similarities between the two species in the shapes, projections and electrophysiology of these neurons suggest that they serve the same functions in both species.These studies were funded by the National Health and Medical Research Council (Australia)     

Characterisation of calcitonin gene-related peptide-immunoreactive neurons in the myenteric plexus of rat colon

A mechanical or chemical stimulus applied to the intestinal mucosa induces motility reflexes in the rat colon. Enteric neurons containing calcitonin gene-related peptide (CGRP) have been suggested as intrinsic primary afferent neurons responsible for mediating such reflexes. In the present study, immunohistochemistry was performed on whole-mount stretch preparations to investigate chemical profiles, morphological characteristics and projections of CGRP-containing neurons in the myenteric plexus of the rat colon. CGRP-positive neuronal cell bodies were detected in preparations incubated with colchicine-containing medium, whereas CGRP-positive nerve fibres were found in colchicine-untreated preparations. These neurons had large oval or round cell bodies that were also immunoreactive for the calcium-binding protein calretinin and neurofilament 200. Myenteric neurons positive for both calretinin and neurofilament 200 had several long processes that emerged from the cell body, consistent with Dogiel type II morphology. Application of the neural tracer DiI to the intestinal mucosa revealed that DiI-labelled myenteric neurons each had an oval or round cell body immunoreactive for calretinin. Thus, CGRP-containing myenteric neurons are Dogiel type II neurons and are immunoreactive for calretinin and neurofilament 200 in the rat colon. These neurons probably project to the intestinal mucosa. This study was supported by a Waseda University Grant for Special Research Projects (2008A-889).     

External adrenergic innervation of the three neuron types of dogiel in the plexus myentericus and the plexus submucosus externus of the porcine small intestine

Summary For the simultaneous demonstration of intramural enteric ganglion cells and the adrenergic nerve fibres in the porcine small intestine a combined histochemical method was developed using a hypertonic solution, the main chemicals of which were glyoxylic acid, Nitro-BT^* and NADH. By means of the enzymatic histochemical method reaction for the NADH-dependent dehydrogenase activity with Nitro-BT as an electron acceptor, the identification of the three neuron types of Dogiel (i.e. type I, type II, type III) was for the first time realized in relation with the glyoxylic acid induced fluorescence (GIF) of the plexus myentericus (Auerbach) and the plexus submucosus externus (Schabadasch). Besides the close topographic relationship between the adrenergic varicose axons on the one hand and the perikarya and dendrites of the multidendritic uniaxonal type I cells characterized by radially oriented short and lamellar dendrites and the multidendritic uniaxonal type III cells, characterized by radially oriented long and tapering dendrites on the other hand, it is striking that for the adendritic multiaxonal type II cells the fluorescent varicose fibres adhere closer to the cell bodies and their processes. In principle, the relation between adrenergic varicose axons and neuron types is identical in plexus myentericus (Auerbach) and plexus submucosus externus (Schabadasch), yet with the exception that in the latter no type I neurons are observed.2,2-Di-p-nitrophenyl-5,5-diphenyl-3,3-(3,3-dimethoxy-4,4-diphenylene) ditetrazolium chloride     

Characterisation of neurons expressing calbindin immunoreactivity in the ileum of the unweaned and mature sheep

We have identified the enteric neuron types expressing immunoreactivity for the calcium-binding protein calbindin D28k (CALB) in cryostat sections and whole-mount preparations of myenteric (MP) and submucosal (SMP) plexuses of sheep ileum. We wished to determine whether CALB-IR in the sheep enteric nervous system was expressed in Dogiel type II cells, as in guinea-pig and rat ileum, and could therefore be used as a marker for intrinsic primary afferent neurons. The neurochemical coding of CALB-containing myenteric and submucosal neurons in ileum of unweaned lamb and mature sheep and its co-localisation with various neural markers was studied immunohistochemically. An antiserum against neuronal nuclear protein (NeuN) failed to detect the entire neuronal population; it was expressed only in 48% of neuron-specific enolase (NSE)-immunoreactive (NSE-IR) neurons. Human neuronal protein appeared to occur in the large majority or all neurons. Almost all CALB-IR neurons were: (1) radially multidendritic; (2) eccentric multidendritic; (3) Dogiel type II. CALB-IR occurred in 20–25% of myenteric and 65–75% of submucosal neurons in lamb and mature sheep, with higher values in mature sheep. Nearly all CALB-IR neurons were common choline acetyltransferase (cChAT)-IR, whereas only about 20% of cChAT-IR somata were CALB-IR. In lamb and mature sheep, 90% of MP CALB-IR neurons were peripheral choline acetyltransferase (pChAT)-IR. In lamb SMP, 80±13% of CALB-IR cells were also pChAT-IR, whereas all those in mature SMP were pChAT-IR. Fewer myenteric CALB-IR neurons exhibited tachykinin (TK) in mature sheep (49%) than in lamb (88%). This was also the case for submucosal ganglia (mature sheep, 63%; lamb, 89%). In lamb MP, 77±7% of CALB-IR cells were NeuN-positive. In mature sheep, 73±10% of CALB-IR somata were NeuN-IR, but NeuN failed to stain SMP neurons. In the MP of suckling and mature sheep, Dogiel type II CALB-IR neurons were calcitonin gene-related peptide (CGRP)-IR. In the SMP at both stages, Dogiel type II CALB-IR somata (about 50% of CALB-IR neurons) were also CGRP-IR. Only small proportions of CALB-IR neurons showed immunoreactivity for calretinin or nitric oxide synthase (NOS), although large populations of CALB and NOS neurons occurred in the ganglia. Thus, CALB is a marker of most Dogiel type II neurons in the sheep but is not confined to Dogiel II neurons. CGRP is a more selective marker of Dogiel type II neurons, being only found in this neuron type.This work was supported by a grant from the Ministero dellIstruzione, dellUniversità e della Ricerca (MIUR)     

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.     

Some neurohistochemical properties of nerve elements in myenteric plexus of rabbit ileum: similarities and dissimilarities to the rodent pattern

Enteric neurons have distinct neurochemical codings in each species. The basal tone of the gastrointestinal tract of the rabbit is low and produces neurally evoked pendular movements. Therefore, it might have an innervation pattern different from that of other laboratory animals. We have characterised myenteric neuron populations in rabbit ileum with neurochemical markers that are known to be associated with distinct cell types and/or fibre systems in the myenteric plexus. The density of nerve cells estimated with the NADH-diaphorase technique was about 2500 cells/cm² and most, if not all, neurons contained microtubule-associated protein 2. NADPH-diaphorase-positive cells were numerous. One cell type was large and emitted long straight processes, whereas small cells bore thin filamentous dendrites. Neurons immunoreactive for 28-kDa calcium-binding protein were rare. Over 70% of them had very strongly labelled lamellar dendrites. Their axons were beaded and formed pericellular baskets around unstained somata. We found very few small tyrosine-hydroxylase-positive cells. The fibre network in the plexus was very strong; the axons formed many pericellular baskets. In double labelling studies, no co-localisation was revealed between the 28-kDa calcium-binding protein and NADPH-diaphorase. Some fibres containing 28-kDa calcium-binding protein formed only a few contacts on somata of NADPH-diaphorase-positive cells. None of the NADPH-diaphorase-labelled cells were found to be stained for tyrosine hydroxylase. Tyrosine-hydroxylase-positive fibres rarely made pericellular baskets on the surface of NADPH-diaphorase-positive somata. Strongly immunolabelled pericellular baskets were never observed around NADPH-diaphorase-positive cell somata. The results suggest that myenteric neurons in rabbit comprise distinct and characteristic neurochemical properties that are different from the rodent pattern. Therefore, the explanation of the motility pattern of rabbit intestine can be approached on a chemical neuroanatomical basis. Received: 6 August 1997/Accepted: 8 October 1997     

Identification of the populations of enteric neurons that have NK1 tachykinin receptors in the guinea-pig small intestine

Simultaneous immunofluorescence labelling was used to investigate the patterns of colocalisation of the NK1 tachykinin receptor with other neuronal markers, and hence determine the functional classes of neuron that bear the NK1 receptor in the guinea-pig ileum. In the myenteric plexus, 85% of NK1 receptor-immunoreactive (NK1r-IR) nerve cells had nitric oxide synthase (NOS) immunoreactivity and the remaining 15% were immunoreactive for choline acetyltransferase (ChAT). Of the latter group, about 50% were immunoreactive for both neuropeptide Y (NPY) and somatostatin (SOM), and had the morphologies of secretomotor neurons. Many of the remaining ChAT neurons were immunoreactive for calbindin or tachykinins (TK), but not both. These calbindin immunoreactive neurons had Dogiel type II morphology. No NK1r-IR nerve cells in the myenteric plexus had serotonin or calretinin immunoreactivity. In the submucosal ganglia, 84% of NK1r-IR nerve cells had neuropeptide Y immunoreactivity and 16% were immunoreactive for TK. It is concluded that NK1r-IR occurs in five classes of neuron; namely, in the majority of NOS-immunoreactive inhibitory motor neurons, in ChAT/TK-immunoreactive excitatory neurons to the circular muscle, in all ChAT/NPY/SOM-immunoreactive secretomotor neurons, in a small proportion of ChAT/calbindin myenteric neurons, and in about 50% of ChAT/TK submucosal neurons.     

Adenylyl cyclase co-distribution with the CaBPs, calbindin-D28 and calretinin, varies with cell type: assessment with the fluorescent dye, BODIPY forskolin, in enteric ganglia

The aims of the present study were: (1) to evaluate BODIPY forskolin as a suitable fluorescent marker for membrane adenylyl cyclase (AC) in living enteric neurons of the guinea-pig ileum; (2) to test the hypothesis that AC is distributed in several subpopulations of enteric neurons; (3) to test the hypothesis that the distribution of AC in the myenteric plexus is not unique to AH/Type 2 neurons. BODIPY forskolin was used to assess the co-distribution of AC in ganglion cells expressing the specific calcium-binding proteins (CaBPs), calretinin, calbindin-D₂₈, and s-100. Cultured cells or tissues were incubated with 10?μM BODIPY forskolin for 30?min and fluorescent labeling was monitored by using laser scanning confocal microscopy. BODIPY forskolin stained the cell soma, neurites, and nerve varicosities of Dogiel Type I or II neurons. About 99% of myenteric and 27% of submucous ganglia contained labeled neurons. About 14% of myenteric and 3% of submucous glia with immunoreactivity for s-100 protein displayed BODIPY forskolin fluorescence. BODIPY forskolin differentially labeled myenteric neurons immunoreactive for calbindin-D₂₈ (80%) and calretinin (17%). The majority (63%) of BODIPY forskolin-labeled myenteric neurons displayed no immunoreactivity for either CaBP. In submucous ganglia, the dye labeled 44.6% of calretinin-immunoreactive neurons, representing 21% of all labeled neurons; it also labeled varicose nerve fibers running along blood vessels. AC thus exists in myenteric Dogiel type II/AH neurons, enteric cholinergic S/Type 1 neurons, and other unidentified non-cholinergic S/Type 1 neurons. Our data also support the hypothesis that AC is expressed in distinct functional subpopulations of AH and S neurons in enteric ganglia, and show that BODIPY forskolin is a suitable marker for AC in immunofluorescence co-distribution studies involving living cells or tissues.     

Quantitative analysis of inputs to somatostatin-immunoreactive descending interneurons in the myenteric plexus of the guinea-pig small intestine

Somatostatin immunoreactivity occurs in a specific subgroup of cholinergic descending interneurons in the myenteric plexus of the guinea-pig small intestine. In the present work, we made light- and electron-microscopic investigations of chemically defined inputs to these neurons, in order that the origins of the connections of other neurons with them could be deduced. Somatostatin-immunoreactive synapses and close contacts were found on the cell bodies and filamentous processes of somatostatin neurons; these were 84% of all inputs. It is thus confirmed that this class of interneuron forms chains that project anally. Descending interneurons with immunoreactivity for nitric oxide synthase provided 14% of inputs to somatostatin-immunoreactive descending interneurons. An antiserum against a calcium-binding protein, calbindin, was used as marker for the majority of intrinsic primary afferent neurons, AH/Dogiel type II neurons; this class of neurons provided only 2.5% of the inputs to somatostatin-immunoreactive descending interneurons. We conclude that somatostatin-immunoreactive descending interneurons are involved in the conduction of impulses distally along the full length of the small intestine, but receive only a minor input from calbindin-immunoreactive primary afferent neurons.     

Platelet-activating factor in the enteric nervous system of the guinea pig small intestine

Platelet-activating factor (PAF) is a proinflammatory mediator that may influence neuronal activity in the enteric nervous system (ENS). Electrophysiology, immunofluorescence, Western blot analysis, and RT-PCR were used to study the action of PAF and the expression of PAF receptor (PAFR) in the ENS. PAFR immunoreactivity (IR) was expressed by 6.9% of the neurons in the myenteric plexus and 14.5% of the neurons in the submucosal plexus in all segments of the guinea pig intestinal tract as determined by double staining with anti-human neuronal protein antibody. PAFR IR was found in 6.1% of the neurons with IR for calbindin, 35.8% of the neurons with IR for neuropeptide Y (NPY), 30.6% of the neurons with IR for choline acetyltransferase (ChAT), and 1.96% of the neurons with IR for vasoactive intestinal peptide (VIP) in the submucosal plexus. PAFR IR was also found in 1.5% of the neurons with IR for calbindin, 51.1% of the neurons with IR for NPY, and 32.9% of the neurons with IR for ChAT in the myenteric plexus. In the submucosal plexus, exposure to PAF (200-600 nM) evoked depolarizing responses (8.2 +/- 3.8 mV) in 12.4% of the neurons with S-type electrophysiological behavior and uniaxonal morphology and in 12.5% of the neurons with AH-type electrophysiological behavior and Dogiel II morphology, whereas in the myenteric preparations, depolarizing responses were elicited by a similar concentration of PAF in 9.5% of the neurons with S-type electrophysiological behavior and uniaxonal morphology and in 12.0% of the neurons with AH-type electrophysiological behavior and Dogiel II morphology. The results suggest that subgroups of secreto- and musculomotor neurons in the submucosal and myenteric plexuses express PAFR. Coexpression of PAFR IR with ChAT IR in the myenteric plexus and ChAT IR and VIP IR in the submucosal plexus suggests that PAF, after release in the inflamed bowel, might act to elevate the excitability of submucosal secretomotor and myenteric musculomotor neurons. Enhanced excitability of motor neurons might lead to a state of neurogenic secretory diarrhea.     

Neurochemical classification of enteric neurons in the guinea-pig distal colon

Previous studies have identified the chemistries, shapes, projections and electrophysiological characteristics of several populations of neurons in the distal colon of the guinea-pig but it is unknown how these characteristics correlate to define the classes of neurons present. We have used double-label immunohistochemical techniques to identify neurochemically distinct subgroups of enteric neurons in this region. On the basis of colocalisation of neurochemical markers and knowledge gained from previous studies of neural projections, 17 classes of neurons were identified. The myenteric plexus contained the cell bodies of 13 distinct types of neurons. Four classes of descending interneurons and three classes of ascending interneurons were identified, together with inhibitory and excitatory motor neurons to both the circular and longitudinal muscle layers. Dogiel type II neurons, which are presumed to be intrinsic primary afferent neurons, were located in myenteric and submucosal ganglia; they were all immunoreactive for choline acetyltransferase and often calbindin and tachykinins. Three classes of secretomotor neurons with cell bodies in submucosal ganglia were defined. Two of these classes were immunoreactive for choline acetyltransferase and the other class was immunoreactive for both vasoactive intestinal peptide and nitric oxide synthase. Some of the secretomotor neurons probably also have a vasomotor function. The neural subtypes defined in the present study are similar in many respects to those found in the small intestine, although differences are evident, especially in populations of interneurons. These differences presumably reflect the differing physiological roles of the two intestinal regions.     

Neurochemically similar myenteric and submucous neurons directly traced to the mucosa of the small intestine

Summary Antisera to neuropeptide Y (NPY) gave an intense immunohistochemical reaction of certain nerve cells in the myenteric and submucous plexuses of the guinea-pig small intestine. Each nerve cell had up to 20 branching, tapering processes that were less than 50 m long and a long process that could be followed for a considerable distance. This morphology corresponds to that of the type-III cells of Dogiel. The long process of each myenteric cell ran through the circular muscle to the submucosa, and in most cases the process could be traced to the mucosa. The submucous nerve cell bodies also had processes that extended to the mucosa. These cell bodies, in both plexuses, also stained with antisera raised against calcitonin generelated peptide (CGRP), cholecystokinin (CCK), choline acetyltransferase (ChAT) and somatostatin (SOM), but did not stain with antibodies against enkephalin, substance P or vasoactive intestinal peptide. Thus, it has been possible for the first time to trace the processes of chemically specified neurons through the layers of the intestinal wall and to show by a direct method that CGRP/CCK/ChAT/NPY/ SOM myenteric and submucous nerves cells provide terminals in the mucosa.     

Characterization of excitatory and inhibitory motor neurons to the human gastric clasp and sling fibers

The aim of this study was to determine the morphology and position of the excitatory and inhibitory motor neurons to the human gastric sling and clasp fibers. Motor neurons were identified by retrograde staining with 1,1'-didodecyl 3,3,3',3'-indocarbocyanine perchlorate (DiI), and choline acetyltransferase (ChAT) or nitric oxide synthase (NOS) immunoreactivity was then determined in these motor neurons. In the sling preparations, 46% of the DiI-stained cells were aboral motor neurons, 43% were local motor neurons, and only 10% were descending motor neurons. Overall, 58% were immunoreactive for ChAT, and 36% for NOS (P = 0.042). Sixty-two percent of local, and 66% of aboral DiI-stained motor neurons were immunoreactive for ChAT. In the clasp preparations, 52% of the DiI-stained cells were descending motor neurons, 45% were local motor neurons, and only 3% were aboral neurons. Overall, 31% were immunoreactive for ChAT and 65% for NOS (P = 0.039). Eighty-five percent of the DiI-stained descending motor neurons were immunoreactive for NOS. All of the cells that were labeled adequately had a single axon and a number of filamentous or flattened lobular dendrites, and fitted into the broad category of Dogiel type I neurons. In conclusion, the majority of the motor neurons to the sling fibers were ChAT-positive excitatory neurons from the myenteric plexus of the stomach and the local region, and to the clasp were predominantly NOS-positive inhibitory neurons from the esophagus.     

Immunohistochemical analysis of substance P-containing neurons in rat small intestine

The neuropeptide substance P (SP) is involved in the regulation of epithelial secretion and motility in the rat small intestine. The morphology, chemical profiles and proportion of SP-containing enteric neurons in this tissue have been examined by immunohistochemical analysis of whole-mount preparations obtained from colchicine-treated rats. In the submucosal plexus of the duodenum, jejunum and ileum, the proportion of SP-positive neurons is 53%, 51% and 49%, respectively. All SP-positive submucosal neurons are positive for neurofilament 200 (NF-200) and calretinin. Immunoreactivity for calcitonin gene-related peptide (CGRP) is detectable in 55% of the SP-positive submucosal neurons. Some SP-positive submucosal neurons have two or more long processes emerging from an oval or round cell body, a characteristic of the Dogiel type II neuron (type II neuron; a putative intrinsic primary afferent neuron). About one-third of the neurons in the myenteric plexus are positive for SP and a majority of them are NF-200/calretinin-positive type II neurons. Immunoreactivity for the SP receptor neurokinin-1 receptor (NK1R) has been detected mainly in the submucosal and myenteric NF-200-positive neurons, which are expected to contain SP. These neurons possibly stimulate each other via SP release. Most of the submucosal and myenteric neurons, including type II neurons, show immunoreactive for the prostaglandin E2 receptor EP3 receptor (EP3R). Thus, SP/NF-200/calretinin/NK1R/EP3R is the common chemical profile of type II neurons in the rat small intestine. The proportion of SP-immunopositive submucosal neurons (49%–53%) is higher in the rat small intestine than in the colon (≤11%) and around 50% are positive for CGRP.     

Morphology of enkephalin-immunoreactive myenteric neurons in the human gut

The aim of this study was the morphological and further chemical characterisation of neurons immunoreactive for leu-enkephalin (leuENK). Ten wholemounts of small and large intestinal segments from nine patients were immunohistochemically triple-stained for leuENK/neurofilament 200 (NF)/substance P (SP). Based on their simultaneous NF-reactivity and 3D reconstruction of single NF-reactive cells, 97.5% of leuENK-positive neurons displayed the appearance of stubby neurons: small somata; short, stubby dendrites and one axon. Of these leuENK-reactive stubby neurons, 91.3% did not display co-reactivity for SP whereas 8.7% were SP-co-reactive. As to their axonal projection pattern, 50.4% of the recorded leuENK stubby neurons had axons running orally whereas in 29.4% they ran anally; the directions of the remaining 20.2% could not be determined. No axons were seen to enter into secondary strands of the myenteric plexus. Somal area measurements revealed clearly smaller somata of leuENK-reactive stubby neurons (between 259±47 m² and 487±113 m²) than those of putative sensory type II neurons (between 700±217 m² and 1,164±396 m²). The ratio dendritic field area per somal area of leuENK-reactive stubby neurons was between 2.0 and 2.8 reflecting their short dendrites. Additionally, we estimated the proportion of leuENK-positive neurons in comparison to the putative whole myenteric neuron population in four leuENK/anti-Hu doublestained wholemounts. This proportion ranged between 5.9% and 8.3%. We suggest leuENK-reactive stubby neurons to be muscle motor neurons and/or ascending interneurons. Furthermore, we explain why we do not use the term Dogiel type I neurons for this population.     

Distribution of P2Y<Subscript>2</Subscript> receptors in the guinea pig enteric nervous system and its coexistence with P2X<Subscript>2</Subscript> and P2X<Subscript>3</Subscript> receptors,neuropeptide Y,nitric oxide synthase and calretinin

The distribution of P2Y₂ receptor-immunoreactive (ir) neurons and fibers and coexistence of P2Y₂ with P2X₂ and P2X₃ receptors, neuropeptide Y (NPY), calretinin (CR), calbindin (CB) and nitric oxide synthase (NOS) was investigated with immunostaining methods. The results showed that P2Y₂-ir neurons and fibers were distributed widely in myenteric and submucous plexuses of the guinea pig stomach corpus, jejunum, ileum and colon. The typical morphology of P2Y₂-ir neurons was a long process with strong positive staining on the same side of the cell body. The P2Y₂-ir neurons could be Dogiel type 1. About 40–60% P2X₃-ir neurons were immunoreactive for P2Y₂ in the myenteric plexus and all the P2X₃-ir neurons expressed the P2Y₂ receptor in the submucosal plexus; almost all the NPY-ir neurons and the majority of CR-ir neurons were also immunoreactive for P2Y₂, especially in the myenteric plexus of the small intestine; no P2Y₂-ir neurons were immunoreactive for P2X₂ receptors, CB and NOS. It is shown for the first time that S type/Dogiel type 1 neurons with fast P2X and slow P2Y receptor-mediated depolarizations could be those neurons expressing both P2Y₂-ir and P2X₃-ir and that they are widely distributed in myenteric and submucosal plexuses of guinea pig gut.     

Ultrastructural studies of the myenteric plexus and smooth muscle in organotypic cultures of the guinea-pig small intestine

External muscle and myenteric plexus from the small intestine of adult guinea-pigs were maintained in vitro for 3 or 6 days. Myenteric neurons and smooth muscle cells from such organotypic cultures were examined at the electron-microscopic level. An intact basal lamina was found around the myenteric ganglia and internodal strands. Neuronal membranes, nuclei and subcellular organelles appeared to be well preserved in cultured tissues and ribosomes were abundant. Dogiel type-II neurons were distinguishable by their elongated electron-dense mitochondria, numerous lysosomes and high densities of ribosomes. Vesiculated nerve profiles contained combinations of differently shaped vesicles. Synaptic membrane specializations were found between vesiculated nerve profiles and nerve processes and cell bodies. The majority of nerve fibres were well preserved in the myenteric ganglia, in internodal strands and in bundles running between circular muscle cells. No detectable changes were found in the ultrastructure of the somata and processes of glial cells. Longitudinal and circular muscle cells from cultured tissue had clearly defined membranes with some close associations with neighbouring muscle cells. Caveolae occurred in rows that ran parallel to the long axis of the muscle cells. These results indicate that the ultrastructural features of enteric neurons and smooth muscle of the guinea-pig small intestine are well preserved in organotypic culture.