Calbindin neurons of the guinea-pig small intestine: quantitative analysis of their numbers and projections

Summary The distribution of nerve cells with immunoreactivity for the calcium-binding protein, calbindin, has been studied in the small intestine of the guinea-pig, and the projections of these neurons have been analysed by tracing their processes and by examining the consequences of nerve lesions. The immunoreactive neurons were numerous in the myenteric ganglia; there were 3500±100 reactive nerve cells per cm² of undistended intestine, which is 30% of all nerve cells. In contrast, reactive nerve cells were extremely rare in submucous ganglia. The myenteric nerve cells were oval in outline and gave rise to several long processes; this morphology corresponds to Dogiel's type-II classification. Processes from the cell bodies were traced through the circular muscle in perforating nerve fibre bundles. Other processes ran circumferentially in the myenteric plexus. Removal of the myenteric plexus, allowing time for subsequent fibre degeneration, showed that reactive nerve fibres in the submucous ganglia and mucosa came from the myenteric cell bodies. Operations to sever longitudinal or circumferential pathways in the myenteric plexus indicated that most reactive nerve terminals in myenteric ganglia arise from myenteric cell bodies whose processes run circumferentially for 1.5 mm, on average. It is deduced that the calbindin-reactive neurons are multipolar sensory neurons, with the sensitive processes in the mucosa and with other processes innervating neurons of the myenteric plexus.     

Connections between neurons of sympathetic ganglia and the myenteric nerve plexus of the mammalian colon

By means of retrograde transport of the fluorescent marker primulin the initial part of the sympathetic innervation of the myenteric nervous plexus of the descending colon has been characterized in cats and guinea pigs. When primulin is injected into the myenteric nervous plexus, marked neurons are revealed in the caudal mesenteric ganglion, in the celiac plexus ganglia, in the sympathetic trunk ganglia. The marked nervous populations of the extramural sympathetic ganglia differ in their form, size, number of neurons and their distribution.     

Immunohistochemical localisation of cholinergic markers in putative intrinsic primary afferent neurons of the guinea-pig small intestine

Antibodies against choline acetyltransferase (ChAT) and the vesicular acetylcholine transporter (VAChT) were used to determine whether neurons that have previously been identified as intrinsic primary afferent neurons in the guinea-pig small intestine have a cholinergic phenotype. Cell bodies of primary afferent neurons in the myenteric plexus were identified by their calbindin immunoreactivity and those in the submucous plexus by immunoreactivity for substance P. High proportions of both were immunoreactive for ChAT, viz. 98% of myenteric calbindin neurons and 99% of submucosal substance P neurons. ChAT immunoreactivity also occurred in all nerve cell bodies immunoreactive for calretinin and substance P in the myenteric plexus, but in only 16% of nerve cells immunoreactive for nitric oxide synthase. VAChT immunoreactivity was in the majority of calbindin-immunoreactive varicosities in the myenteric ganglia, submucous ganglia and mucosa and also in the majority of the varicosities of neurons that were immunoreactive for calretinin and somatostatin and that had been previously established as being cholinergic. We conclude that the intrinsic primary afferent neurons are cholinergic and that they may release transmitter from their sensory endings in the mucosa.     

Patch clamp recording from enteric neurons in situ

The study of enteric neurons is key to understanding intestinal motility anGutn of therapeutic strategies for dealing with neurogenic disorders. However, enteric neurons have historically been inaccessible to patch-clamp recording. We report here the first technique that allows patch-clamp recording of neurons from the intact myenteric plexus of the mouse duodenum. The mucosa, submucosa and circular muscles are removed, exposing the myenteric plexus on the longitudinal muscle. Proteolytic treatment of exposed ganglia combined with gentle cell-surface cleaning allows gigaseal formation. Compared with previous studies using intracellular microelectrode recordings or cultured myenteric neurons, this technique provides an opportunity to explore properties of single or multiple ion channels in myenteric neurons in their native environment. The protocol-from the tissue preparation to patch-clamp recording-can be completed in ～4 h.     

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.     

Ultrastructure of blood capillaries in vascular plexuses of the lateral ventricles of the human brain

Ultrastructure of the wall of the microcirculatory bed links in the lateral ventricles of the human brain has been studied, as well as their interrelations with neural elements and ependyma. Together with typical morphological structural signs, certain peculiarities are revealed, characterizing organic specificity. Elements, performing function of the blood-brain barrier are determined: epithelium (ependyma), basal membranes, interstitium. A well developed afferent and efferent nervous apparatus of the vascular plexus, evidently, actively participates in regulation of the microcirculatory blood bed and in formation of liquor.     

Peptide-containing neurons in explant cultures of guinea-pig myenteric plexus during development in vitro: Gross morphology and growth patterns

Summary The gross morphology and growth patterns of substance P, enkephalin-, somatostatin and vasoactive intestinal peptide-immunoreactive neurons have been studied in explant cultures of the myenteric plexus taken from beneath the newborn guinea-pig taenia coli, grown for up to 4 weeks in vitro. Substance P and enkephalin-immuno-reactive neurons were more abundant than somatostatin and vasoactive intestinal peptide-immunoreactive neurons. The peptide-containing neuronal cell bodies were clearly visible in culture and exhibited characteristic gross morphologies similar to those described in situ, although some overlap of shape between populations containing different peptides was seen. All four types of peptide-containing fibres were found in the outgrowth and central areas of the cultures. In the case of substance P and somatostatin, the density and pattern of labelling in the central, neuronal area of the cultures resembled that previously seen in the myenteric plexus of the newborn guinea-pig caecum in situ, while the density of the enkephalin-immunoreactive fibres was greater, and that of the vasoactive intestinal peptide-immunoreactive fibres less than that seen in situ. These observations suggest that subpopulations of myenteric neurons containing different peptides may be differentially affected by the culture environment. Possible contributory factors are discussed.     

Effects of narcotic opiates and serotonin on the electrical behavior of neurons in the guinea pig myenteric plexus

Action potentials were recorded extracellularly from spontaneously firing neurons in the myenteric plexus of the guinea pig ileum. Morphine, which inhibits acetylcholine release from the myenteric plexus, inhibited the spontaneous electrical activity of about half the cells studied, while serotonin elevated the firing rate of these cells. Units not stimulated by serotonin were not inhibited by morphine or levorphanol. Morphine also prevented the increase in firing rate caused by serotonin. These effects of morphine were stereospecific and blocked by naloxone, and are therefore considered to be specific opiate effects. This study demonstrates opposing effects of narcotic opiates and serotonin on the electrical activity of serotoninoceptive neurons in the myenteric plexus.     

大鼠回肠肌间神经丛一氧化氮合酶和乙酰胆碱酯酶双重显示法研究

本文用一氧化氮合酶和乙酰胆碱酯酶双重显示法,对大鼠回肠肌间神经丛进行了组织化学观察,结果发现三种不同染色的神经元：（１）乙酰胆碱酯酶阳性神经元（占８２％）;（２）一氧化氮合酶阳性神经元（占１６％）;（３）一氧化氮合酶和乙酰胆碱酯酶阳性神经元（占２％）。以上结果提示,一氧化氮可以与乙酰胆碱共存于大鼠回肠肌间神经丛的少数神经元内。本文还对肠肌间神经丛内神经元的类型和一氧化氮的作用进行了讨论。     

Distribution and projections of neurons with immunoreactivity for both gastrin-releasing peptide and bombesin in the guinea-pig small intestine

Summary Bombesin-like and gastrin-releasing peptide (GRP)-like immunoreactivities were localized in nerves of the guinea-pig small intestine and celiac ganglion with the use of antibodies raised against the synthetic peptides. The anti-bombesin serum (preincubated to avoid cross reactivity with substance P) and the anti-GRP serum revealed the same population of neurons. Preincubation of the antibombesin serum with bombesin abolished the immunoreactivity in nerves while absorption of the anti-GRP serum with either bombesin or the 14–27 C-terminal of GRP only reduced the immunoreactivity. The immunoreactivity was abolished by incubation with GRP 1–27.Immunoreactive nerves were found in the myenteric plexus, circular muscle, submucous plexus and in the celiac ganglion. Faintly reactive nerve cell bodies were found in the myenteric ganglia (3.2% of all neurons) but not in submucous ganglia. After all ascending and descending pathways in the myenteric plexus had been cut, reactive terminals disappeared in the myenteric plexus, circular muscle (including the deep muscular plexus) and the submucous plexus on the anal side. After the mesenteric nerves were cut no changes were observed in the intestinal wall but the reactive fibres in celiac ganglia disappeared. It is deduced that GRP/bombesin-immunoreactive nerve cell bodies in myenteric ganglia project from the myenteric plexus to other myenteric ganglia situated further anally (average length 12 mm), anally to the circular muscle (average length 9 mm), anally to submucous ganglia (average length 13 mm) and external to the intestine to the celiac ganglia.It is concluded that the GRP/bombesin-reactive neurons in the intestinal wall represent a distinct population of enteric neurons likely to be involved in controlling motility and in the coordination of other intestinal functions.     

Nitric oxide synthase in the enteric nervous system of the guinea-pig: a quantitative description

The distribution and abundance of nitric oxide synthase (NOS)-containing neurons and their terminals in the gastrointestinal tract of the guinea-pig were examined in detail using NADPH diaphorase histochemistry and NOS immunohistochemistry. NOS-containing cell bodies were found in the myenteric plexus throughout the gastrointestinal tract and in the submucous plexus of the stomach, colon and rectum. NOS-containing neurons comprised between 12% (in the duodenum) and 54% (in the esophagus) of total myenteric neurons. In the ileum, NOS neurons represented 19% of total myenteric neurons. Most of the NOS neurons throughout the gastrointestinal tract possessed lamellar dendrites and a single axon. NOS-containing terminals were abundant in the circular muscle, including that of the sphincters, but were rare in the longitudinal muscle, except for the taeniae of the caecum. The muscularis mucosae of the esophagus, stomach, colon and rectum received a medium to dense innervation by NOS terminals. Within myenteric ganglia, NOS-containing terminals were extremely sparse in the esophagus, stomach and duodenum, common in the ileum and distal colon and extremely dense in the proximal colon and rectum. The submucous plexus in the ileum and large intestine contained a sparse plexus of NOS-containing terminals. NOS terminals were not observed in the mucosa of any region. We conclude that throughout the gastrointestinal tract of the guinea-pig, NOS neurons are inhibitory motor neurons to the circular muscle; in the ileum and large intestine, NOS neurons may also function as interneurons.     

Calretinin-immunoreactive neurons and their projections in the guinea-pig colon

The distribution of nerve cells and fibres with immunoreactivity for the calcium-binding protein, calretinin, was studied in the distal colon of the guinea-pig. The projections of the neurons were determined by examining the consequences of lesioning the myenteric plexus. Calretinin-immunoreactive neurons comprised 17% of myenteric nerve cells and 6% of submucous nerve cells. Numerous calretinin-immunoreactive nerve fibres were located in the longitudinal and circular muscle, and within the ganglia of the myenteric and submucous plexuses. Occasional fibres were found in the muscularis mucosae, but they were very rare in the lamina propria of the mucosa. Lesion studies revealed that myenteric neurons innervated the underlying circular muscle and provided both ascending and descending processes that gave rise to varicose branches in myenteric ganglia. Calretinin-immunoreactive fibres also projected to the tertiary component of the myenteric plexus, and are therefore likely to be motor neurons to the longitudinal muscle. Varicose fibres that supplied the submucous ganglia appear to arise from submucous nerve cells. Arterioles of the submucous plexus were sparsely innervated by calretinin-immunoreactive fibres. The submucous plexus was the principal source of immunoreactive nerve fibres in the muscularis mucosae. This work shows that calretinin-IR reveals different neuronal populations in the large intestine to those previously reported in the small intestine.     

Transient expression of neuronal nitric oxide synthase by neurons of the submucous plexus of the mouse small intestine

Although neurons containing neuronal nitric oxide synthase (NOS) are abundant in the myenteric plexus of the small intestine of all mammalian species examined to date, NOS-containing neurons are sparse in the submucous plexus, and there does not appear to be an innervation of the mucosa by nerve fibres containing NOS. In this study, we used immunohistochemical techniques to examine the presence of neuronal NOS in the mouse intestine during development. At embryonic day 18 and postnatal day 0 (P0), about 50% of the neurons in the submucous plexus of the small intestine showed strong immunoreactivity to NOS, and NOS-immunoreactive nerve fibres were present in the mucosa. By P7, there was a gradation in the intensity of NOS immunostaining exhibited by submucosal neurons, varying from intense to extremely weak. During subsequent development, the proportion of submucous neurons showing NOS immunoreactivity decreased, and immunoreactive nerve fibres were no longer observed in the mucosa. In adult mice, NOS neurons comprised only 3% of neurons in the submucous plexus, which is significantly less than at P0. In contrast to the submucous plexus, the percentage of neurons that showed NOS immunoreactivity in the myenteric plexus did not change significantly during development.     

Occurrence,distribution and neurochemical features of small intestinal neurons projecting to the cranial mesenteric ganglion in the pig

The small intestine of the pig has been investigated for its topographical distribution of enteric neurons projecting to the cranial mesenteric ganglion, by using Fast Blue or Fluorogold as a retrogradely transported neuronal tracer. Contrary to the situation in small laboratory animals such as rat and guinea-pig, the intestinofugally projecting neurons in the porcine small intestine were not restricted to the myenteric plexus, but were observed in greater numbers in ganglia of the outer submucous plexus. The inner submucous plexus was devoid of labelled neurons. Retrogradely labelled neurons were mostly found, either singly or in small aggregates, in ganglia located within a narrow border on either side of the mesenteric attachment. For both nerve networks, their number increased from duodenum to ileum. All the retrogradely labelled neurons exhibited a multidendritic uniaxonal appearance. Some of them displayed type-III morphology and stained for serotonin. This study indicates that, in the pig, not only the myenteric plexus but also one submucous nerve network is involved in the afferent component of intestino-sympathico-intestinal reflex pathways. The finding that some of the morphologically defined type-III neurons participate in these reflexes is in accord with the earlier proposal that type-III neurons are supposed to fulfill an interneuronal role, whether intra- or extramurally.     

Divergent fate and origin of neurosphere-like bodies from different layers of the gut

Enteric neural stem cells (ENSCs) are a population of neural crest-derived multipotent stem cells present in postnatal gut that may play an important role in regeneration of the enteric nervous system. In most studies, these cells have been isolated from the layer of the gut containing the myenteric plexus. However, a recent report demonstrated that neurosphere-like bodies (NLBs) containing ENSCs could be isolated from mucosal biopsy specimens from children, suggesting that ENSCs are present in multiple layers of the gut. The aim of our study was to assess whether NLBs isolated from layers of gut containing either myenteric or submucosal plexus are equivalent. We divided the mouse small intestine into two layers, one containing myenteric plexus and the other submucosal plexus, and assessed for NLB formation. Differences in NLB density, proliferation, apoptosis, neural crest origin, and phenotype were investigated. NLBs isolated from the myenteric plexus layer were present at a higher density and demonstrated greater proliferation, lower apoptosis, and higher expression of nestin, p75, Sox10, and Ret than those from submucosal plexus. Additionally, they contained a higher percentage of neural crest-derived cells (99.4 ± 1.5 vs. 0.7 ± 1.19% of Wnt1-cre:tdTomato cells; P < 0.0001) and produced more neurons and glial cells than those from submucosal plexus. NLBs from the submucosal plexus layer expressed higher CD34 and produced more smooth muscle-like cells. NLBs from the myenteric plexus layer contain more neural crest-derived ENSCs while those from submucosal plexus appear more heterogeneous, likely containing a population of mesenchymal stem cells.     

Morphological characteristics of nerves of the vascular plexuses in the lateral cerebral ventricles of human fetuses

The vascular plexus of the lateral ventricle of the human brain includes the microcirculatory bed responsible for the blood supply and liquor production of the organ. Blood capillaries form a thick parviansiform network of the plexus. Innervation of the vascular plexus of the cerebral lateral ventricle in human embryos and fetuses develops synchronously with formation of the plexus itself. The structural complexity of the nervous apparatus increases at the expense of increasing number of nervous fasciculi and single nervous fibers. By the time of birth the nervous apparatus of the plexus acquires a definitive character. After birth further improvement of the nervous apparatus takes place. The role of the microcirculatory bed and of the nervous apparatus of the vascular plexus in ensuring functional loadings and their participation in reactions to certain pathological effects is substantiated.     

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.     

Distribution of neurons with high-affinity uptake sites for GABA in the myenteric plexus of the guinea-pig,rat and chicken

The occurrence of neurons possessing high-affinity uptake sites for GABA was studied in the myenteric plexus of the guinea-pig ileum, caecum, and proximal and distal colon, the rat proximal colon, and the chicken gizzard with the use of 3H-GABA and autoradiography. Experiments were carried out on plexuses that had been freshly isolated from the gut wall or on isolated plexuses that had been maintained as explant cultures for 7 to 14 days. Scattered neurons selectively labelled with 3H-GABA were found in the myenteric plexuses from all the areas examined. The results suggest that GABAergic neurons are widely distributed in the enteric nervous system.     

CCK-A receptor activation induces fos expression in myenteric neurons of rat small intestine

Cholecystokinin (CCK), a hormone secreted from endocrine cells of the small intestine, participates in the control of motility and secretion in the gastrointestinal tract, and in the control of food intake. At least some of the effects of CCK on intestinal function appear to be mediated via activation of intrinsic neurons in the myenteric plexus. However, the distribution of CCK-responsive enteric neurons within the rat small intestine is not known. Neither has the role of CCK-A receptors in the activation of rat myenteric neurons been investigated. Therefore, to determine the distribution of CCK-responsive neurons in the small intestinal myenteric plexus we utilized immunohistochemical detection of Fos, the protein product of the immediate early gene c-fos, to identify neurons that were activated by exogenous CCK. We also monitored Fos expression in the dorsal hindbrain, and examined CCK-induced Fos expression in the presence or absence of a receptor antagonist for the type-A CCK receptor. We found that CCK significantly increased Fos expression in the hindbrain and in myenteric neurons of the duodenum and jejunum, but not the ileum. Neuronal Fos responsiveness in both brain and myenteric neurons was mediated by CCK-A receptors, as CCK-induced Fos expression was eliminated in rats pretreated with a CCK-A receptor antagonist. We conclude that CCK activates small intestinal myenteric neurons, via CCK-A receptors. Activation of these intrinsic intestinal neurons may participate in reflexes and behaviors that are mediated by CCK.