Morphological and immunohistochemical identification of neurons and their targets in the guinea-pig duodenum

Nerve circuits within the proximal duodenum were investigated using a combination of immunohistochemistry for individual neuron markers and lesion of intrinsic nerve pathways to determine axon projections. Cell shapes and axonal projections were also studied in cells that had been injected with a marker substance. Several major neuron populations were identified. Calbindin immunoreactivity occurred in a population of myenteric nerve cells with Dogiel type II morphology. These had axons that projected to other myenteric ganglia, to the circular muscle and to the mucosa. All were immunoreactive for the synthesizing enzyme for acetylcholine, choline acetyltransferase, and some were also immunoreactive for calretinin. Myenteric neurons with nitric oxide synthase immunoreactivity projected anally to the circular muscle. These were also immunoreactive for vasoactive intestinal peptide, and proportions of them had enkephalin and/or neuropeptide Y immunoreactivity. It is suggested that they are inhibitory motor neurons to the circular muscle. A very few (about 2%) of nitric oxide synthase-immunoreactive neurons had choline acetyltransferase immunoreactivity. Tachykinin (substance P)-immunoreactive nerve cells were numerous in the myenteric plexus. Some of these projected orally to the circular muscle and are concluded to be excitatory motor neurons. Others projected to the tertiary plexus which innervates the longitudinal muscle and others provided terminals in the myenteric plexus. Two groups of descending interneurons were identified, one with somatostatin immunoreactivity and one with vasoactive intestinal peptide immunoreactivity. The two most common nerve cells in submucous ganglia were neuropeptide Y- and vasoactive intestinal peptide-immunoreactive nerve cells. Both provided innervation of the mucosa. There was also a population of calretinin-immunoreactive submucous neurons that innervated the mucosal glands, but not the villi. Comparison with the ileum reveals similarities in the chemistries and projections of neurons. Differences include the almost complete absence of nitric oxide synthase immunoreactivity from vasoactive intestinal peptide-immunoreactive interneurons in the duodenum, the projection of calbindin-immunoreactive Dogiel type II neurons to the circular muscle and the absence of tachykinin-immunoreactivity from these neurons.     

The neurochemical coding and projections of circular muscle motor neurons in the human colon

BACKGROUND & AIMS: Enteric neurons can be characterized by their chemical coding, projections, and morphology. The aim of this study was to describe the different classes of human colonic circular muscle motor neurons. METHODS: Human colonic circular muscle motor neurons were identified by retrograde tracing with 1,1'-didodecyl 3,3,3',3'-indocarbocyanine perchlorate (Dil) applied to the circular muscle layer. Whole-mount preparations of the myenteric plexus were then double-labeled with antisera to choline acetyltransferase (ChAT) and/or nitric oxide synthase (NOS), or NOS and vasoactive intestinal peptide (VIP), and the position and immunoreactivity of Dil-filled neurons were recorded. RESULTS: Fifty-two percent of all Dil-filled neurons were ChAT immunoreactive, and 86% of these projected up to 11 mm orally, with 14% projecting short distances anally. Forty-eight percent of the Dil-filled neurons were NOS immunoreactive, and 77% of these projected up to 19 mm anally, with 23% projecting no more than 6 mm orally. A subpopulation of these NOS-immunoreactive motor neurons were also VIP-immunoreactive. A small population of myenteric neurons was immunoreactive for both ChAT and NOS, but none projected to the circular muscle. NOS-immunoreactive motor neurons projected for longer distances than those with ChAT immunoreactivity and were larger. CONCLUSIONS: There are two classes of human colonic motor neurons: one is excitatory (ChAT-immunoreactive) and mainly projects orally and the other is inhibitory (NOS +/- VIP immunoreactive) and projects preferentially anally.     

Insulin-stimulated cell growth in insulin receptor substrate-1-deficient ZR-75-1 cells is mediated by a phosphatidylinositol-3-kinase-independent pathway

The projections of enteric neurons to the circular muscle of the guinea pig gastric corpus were investigated systematically by using the retrogradely transported fluorescent carbocyanine dye 1,1'-didodecyl-3,3,3',3'-tetramethyl indocarbocyanine perchlorate (DiI), applied to the muscle layer or myenteric plexus in vitro. DiI-labeled motor neuron cell bodies were located up to 6.3 mm aboral, 17 mm oral, and up to 20 mm circumferential to the DiI application site. Labeled nerve fibers ran for long distances from the DiI application site toward the greater and lesser curvatures, where they coursed parallel to the bundles of the "gastric sling" muscle. The majority of labeled cells were located toward the lesser curvature of the stomach. Nerve cell bodies that were aboral to the DiI application site were usually small, immunoreactive for choline acetyltransferase, and, thus, were likely to be excitatory motor neurons. Neurons that were located orally were larger, fewer in number, and immunoreactive for nitric oxide synthase and, thus, were likely to be inhibitory motor neurons. Application of DiI directly to the myenteric plexus filled neurons up to 15 mm aborally and up to 21 mm orally but labeled few neurons circumferentially. All nerve cells that were filled from either the circular muscle or the myenteric plexus had Dogiel type I morphological features. These results demonstrate a clear polarity of projection of inhibitory and excitatory motor neurons and a functionally continuous innervation of the circular and gastric sling muscle layers. Nonmotor neurons in the myenteric plexus were demonstrated, but neurons with Dogiel type II morphological features are apparently absent.     

Extrinsic denervation increases myenteric nitric oxide synthase-containing neurons and inhibitory neuromuscular transmission in guinea pig

Enteric nerves can function normally without connections with the central nervous system. A contributing component of the functional autonomy exhibited by enteric nerves is their plasticity. In the present study, the number of nitric oxide synthase-immunoreactive (NOS-ir) myenteric neurons and inhibitory neuromuscular transmission were studied in extrinsically denervated ileal segments. Segments of ileum were extrinsically denervated by crushing the mesenteric blood vessels supplying a loop of ileum in anesthetized guinea pigs. Some unoperated animals were treated with capsaicin or 6-hydroxydopamine (6-OHDA) to disrupt primary afferent and sympathetic nerves, respectively. NOS-ir was localized using indirect immunofluorescence. Nerve-mediated relaxations of longitudinal muscle were studied in vitro using standard methods. At 7 weeks after extrinsic denervation there was a 93% increase in the number of NOS-ir myenteric neurons. The number of neurons containing detectable vasoactive intestinal peptide-ir neurons was not changed after extrinsic denervation. Neurogenic relaxations caused by 10, 20 and 50 Hz transmural stimulation were larger in extrinsically-denervated tissues compared to control tissues. The NOS antagonist, nitro-L-arginine (300 microM) inhibited neurogenic relaxations in control and extrinsically-denervated tissues. Capsaicin- but not 6-OHDA-treatment mimicked the effects of extrinsic denervation on NOS-ir and neurogenic relaxations of the longitudinal muscle. Active or passive properties of the longitudinal muscle were unaffected by extrinsic denervation. These data indicate that extrinsic denervation is associated with an increase in the number of myenteric neurons expressing detectable NOS-ir and potentiation of inhibitory transmission to longitudinal muscle. This effect is due to loss of extrinsic sensory nerves.     

Oxygen sensing by the global regulator, FNR: the role of the iron-sulfur cluster

Pituitary adenylate cyclase-activating peptide (PACAP)-immunoreactive (IR) neurons in the myenteric and submucosal plexus of the rat small and large intestine were examined by immunostaining with purified polyclonal antiserum against PACAP (1-15), using both light and electron microscopy. Many PACAP-IR neuronal cell bodies and fibers were found in the myenteric and submucosal plexus. Many of the PACAP-IR fibers originated from the cell bodies of the myenteric and submucosal ganglia. The ganglia were also innervated by PACAP-IR fibers. PACAP-IR fibers penetrated both the circular and longitudinal muscle layers, confirming the previous observations indicating that PACAP neurons act as motor neurons. Ultrastructural study demonstrated that PACAP-IR nerve terminals formed synaptic contacts with PACAP-IR nerve cell bodies or dendritic processes. This observation suggests that PACAP-IR neurons innervate other PACAP-IR neurons, and that PACAP neurons work as interneurons in the enteric nervous system. PACAP-IR nerve cells received not only PACAP-positive nerve terminal input also PACAP-negative nerve terminal input. It also suggests that PACAP neurons are regulated not only by PACAP-IR enteric neurons, but also by neurons originating elsewhere. Our observations support the view that PACAP-IR neurons are involved in the control of gut motility.     

Projections of pelvic autonomic neurons within the lower bowel of the male rat: an anterograde labelling study

The tissues of the large intestine which receive an innervation by neurons of the major pelvic ganglia were identified following in vivo and in vitro anterograde labelling with the lipophilic tracer 1,1'didodecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate in the male rat. The primary target in the gut of major pelvic ganglion neurons is the myenteric plexus of the distal colon and the rectum. The serosal ganglia, on the surface of the most distal region of the rectum and the circular muscle of the distal colon and rectum were less densely innervated. The pelvic ganglia do not innervate the longitudinal muscle, submucosal blood vessels, submucosal plexus, or mucosa. The pelvic supply reaches the bowel via two groups of rectal nerves and branches of the penile nerves. All of these connections also carry the axons of viscerofugal neurons from the bowel, some of which have terminal axons in the major pelvic ganglia. Finally, the different nerves supplied different targets. In particular, while the rectal nerves carried pelvic axons supplying the myenteric plexus, circular muscle, and serosal ganglia, the penile nerves only innervated the serosal ganglia. In addition, the two groups of rectal nerves innervated slightly different regions of the bowel and provided different projection patterns. However, successful in vivo labelling was achieved in only 6/12 animals and while all in vitro experiments resulted in successful labelling, it was clear that only a proportion of pelvic projections in any given nerve were labelled. These studies have shown that the major pelvic ganglia are primarily involved in the control of motility, but not of vascular and secretomotor functions. Thus pelvic neurons do not innervate the same range of target tissues within the bowel as the prevertebral ganglia. This study has also shown that the different pathways to the gut from the major pelvic ganglia innervate different tissues, suggesting that the autonomic innervation of the gut is not homogeneous along its length.     

Vagal efferent and afferent innervation of the rat esophagus as demonstrated by anterograde DiI and DiA tracing: focus on myenteric ganglia

Anterograde tracing with the carbocyanine tracer DiI and the aminostyrol derivative DiA was used to selectively label fibers from the nucleus ambiguus, dorsal motor nucleus and nodose ganglion, respectively, terminating in the rat esophagus, and to compare them with the innervation of the gastric fundus in the same animals. Ambiguus neurons terminated on motor endplates distributed mainly to the ipsilateral half of the esophagus. There was no evidence of preganglionic innervation of myenteric ganglia from ambiguus neurons. Neurons of the dorsal motor nucleus supplied sparse fibers to only about 10% of enteric ganglia in the esophagus while they innervated up to 100% of myenteric ganglia in the stomach. Neurons of the nodose ganglion terminated profusely on more than 90% of myenteric ganglia of the esophagus and on about 50% of ganglia in the stomach. Afferent vagal fibers were also frequently found in smooth muscle layers starting at the esophago-gastric junction. In contrast, they were extremely rare in the striated muscle part of the esophagus. These morphological data suggest a minor influence of neurons of the dorsal motor nucleus and a prominent influence of vagal afferent terminals onto myenteric neurons in the rat esophagus.     

Functional changes in nonadrenergic, noncholinergic inhibitory neurons in ileal circular smooth muscle after small bowel transplantation in rats

This experiment was designed to determine mechanisms of change in nonadrenergic, noncholinergic (NANC) inhibitory neurons in the ileum after small bowel transplantation (SBT) in the rat and whether nitric oxide (NO) serves as an important NANC inhibitory neurotransmitter in the rat ileum. Eight groups of rats (N > or =8 rats/group) were studied: neurally intact unoperated controls; rats one week after anesthesia and sham celiotomy; and separate groups one and eight weeks after either 40 min of cold ischemia of the jejunoileum, combined jejunal and ileal intestinal transection/reanastomosis, or orthotopic SBT of the entire jejunoileum. Contractile activity was evaluated in full-thickness ileal circular muscle strips under isometric conditions. Spontaneous activity did not differ among groups. In all groups, exogenous NO, NG-monomethyl-L-arginine (L-NMMA, an NO synthase inhibitor), and methylene blue (soluble guanylate cyclase inhibitor) had no effect on spontaneous activity, while 8-bromocyclic guanosine monophosphate (8Br-cGMP) inhibited contractile activity in all groups. Low frequency (2-10 Hz) electrical field stimulation (EFS) inhibited contractile activity only in control and SBT groups; L-NMMA and methylene blue did not alter the response to EFS in any group. These results suggest that each aspect of the SBT procedure, ischemia/reperfusion injury, disruption of enteric neural continuity by intestinal transection, and extrinsic denervation, alter function of enteric ileal inhibitory neurons separately early (one week) after operation. NO, a known inhibitory neurotransmitter in other gut regions, does not affect ileal circular muscle in neurally intact tissue nor mediate functional changes in inhibitory nerve function nor smooth muscle contractility after SBT.     

Action of substance P and interaction of calcitonin gene-related peptide and substance P on the cat antral circular muscle

The actions of substance P (SP) and calcitonin gene-related peptide (CGRP) and their interaction were examined in vitro in the feline antrum and colon. Circular muscle contraction was seen in the antrum to both peptides, but only to SP in the proximal colon. Antral contraction was enhanced when both peptides were given together. This interaction was inhibited by tetrodotoxin or atropine. SP acted at the antrum via a smooth muscle neurokinin receptor which is not a (NK)-1 receptor. SP binding was displaced by neurokinin A but not by the NK-1 receptor antagonist, CP-96345. The colonic response was inhibited by CP-96345. Immunohistochemistry revealed SP-like immunoreactivity (SP-LI) in fibers in the antral myenteric plexus and circular muscle, while CGRP-like immunoreactivity (CGRP-LI) was seen in the myenteric plexus only, without co-localization. These studies supported the hypothesis that SP acted via the NK-2 receptor at the feline circular muscle in the antrum to induce contraction and at the NK-1 receptor in the proximal colon. CGRP enhanced the effect of SP via a cholinergic pathway.     

Detection of naturally expressed receptors for gastrin-releasing peptide and tachykinins using cyanine 3-labelled neuropeptides

Peptides labelled with the fluorophore cyanine 3 were used to study naturally expressed neuropeptide receptors by confocal microscopy in continuous cell lines, primary cultures, and unfixed tissue. Swiss 3T3 fibroblasts bound cyanine 3-gastrin-releasing peptide at 4 degrees C, and internalized the peptide after 10 min at 37 degrees C. Internalization was specific, since it was blocked by incubation with unlabelled peptide. Primary cultures of myenteric neurons of the guinea pig incubated with cyanine 3-substance P at 4 degrees C had specific surface labelling. After 30 s at 37 degrees C, the peptide was internalized into vesicles in both the soma and neurites. Direct observation of live neurons showed movement of fluorescent vesicles to a perinuclear region after 30 min. Endocytosis was associated with a loss of surface binding sites. Unfixed whole mounts of guinea pig and rat ileum were incubated with cyanine 3-neurokinin A at 4 degrees C. After 5 min at 37 degrees C, Cy3-neurokinin A was specifically internalized in neurons and smooth muscle cells. After 30 min, a perinuclear labelling occurred in some cells. Labelling in rat neurons was diminished by the NK3-R antagonist SR142801. Thus, cyanine 3-neuropeptides are valuable tools to study expression and endocytosis of naturally expressed receptors.     

Neurochemical classification of myenteric neurons in the guinea-pig ileum

A strategy has been developed to identify and quantify the different neurochemical populations of myenteric neurons in the guinea-pig ileum using double-labelling fluorescence immunohistochemistry of whole-mount preparations. First, six histochemical markers were used to identify exclusive, non-overlapping populations of nerve cell bodies. They included immunoreactivity for the calcium binding proteins calbindin and calretinin, the neuropeptides vasoactive intestinal polypeptide, substance P and somatostatin, and the amine, 5-hydroxytryptamine. The sizes of these populations of neurons were established directly or indirectly in double-labelling experiments using a marker for all nerve cell bodies. Each of these exclusive populations was further subdivided into classes by other markers, including immunoreactivity for enkephalins and neurofilament protein triplet. The size of each class was then established directly or by calculation. These distinct, neurochemically-identified classes were related to other published work on the histochemistry, electrophysiology and retrograde labelling of enteric neurons and to the simple Dogiel morphological classification. A classification scheme, consistent with previous studies, is proposed. It includes 14 distinct classes of myenteric neurons and accounts for nearly all neurons in the myenteric plexus of the guinea-pig ileum.     

Purinergic fast excitatory postsynaptic potentials in myenteric neurons of guinea pig: distribution and pharmacology

BACKGROUND & AIMS: Adenosine triphosphate (ATP) acting at P2 receptors mediates some fast excitatory postsynaptic potentials (fEPSPs) in myenteric neurons of guinea pig ileum. The present studies investigate the distribution of purinergic fEPSPs along the length of the gut and characterize the P2-receptor subtype mediating fEPSPs. METHODS: Conventional intracellular electrophysiological methods were used to record from myenteric neurons in vitro. RESULTS: At a membrane potential of -97 +/- 1 mV, the amplitude (25 +/- 1 mV; n = 307) of fEPSPs was similar along the gut. Hexamethonium (100 micromol/L) inhibited fEPSPs in the gastric corpus by 98% +/- 1% (n = 31) and in the duodenum, ileum, taenia coli, proximal colon, and distal colon by 42%-55%. In the presence of hexamethonium, suramin (100 micromol/L) or the P2X antagonist pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS, 10 micromol/L) reduced the control fEPSP amplitude in the duodenum, ileum, taenia coli, proximal colon, and distal colon by 71%-84%. The pharmacology of the purinergic fEPSPs was investigated in detail in the ileum. Noncholinergic fEPSPs were concentration-dependently (1-30 micromol/L) inhibited by PPADS (50%-inhibitory concentration, 3 micromol/L). In addition, alpha,beta-methylene 5'-adenosine triphosphate (1 micromol/L) also reduced purinergic fEPSPs. CONCLUSIONS: Fast EPSPs mediated in part through P2X receptors are prominent in myenteric neurons along the small and large intestines but are rare in the gastric corpus.     

Characterization and mediation of inhibitory junction potentials from opossum lower esophageal sphincter

BACKGROUND: Activating nonadrenergic, noncholinergic (NANC) nerves of the lower esophageal sphincter (LES) hyperpolarizes and relaxes its circular smooth muscle. This relaxation is mediated by nitric oxide (NO) or an NO-containing compound. These studies were undertaken to compare the electrophysiological responses of circular smooth muscle from the LES and esophagus in response to NANC nerve stimulation and to test the hypothesis that NO mediates LES hyperpolarization. METHODS: The transmembrane potential difference was recorded with glass microelectrodes. Nerve-mediated membrane responses were evoked by electrical pulses of 0.5 msec duration and 50 V amplitude. RESULTS: Responses of LES muscle differed from those of the esophageal muscle. The duration of hyperpolarization was much longer in sphincteric muscle. The depolarization that followed hyperpolarization of esophageal muscle was not observed in sphincteric muscle. NG-nitro-L-arginine, an inhibitor of NO synthase, attenuated the nerve-induced hyperpolarization. L-arginine, the substrate for NO synthase, antagonized the effect of NG-nitro-L-arginine. Exogenous NO hyperpolarized of the smooth muscle membrane. CONCLUSIONS: These data support the hypothesis that NO or an NO-like compound may mediate nerve-induced hyperpolarization of the opossum LES.     

The effects of heating with ultrasound on knee joint displacement

The effect of prostaglandin E2 (PGE2) on the activity-related expression of the proto-oncogene c-fos in specific populations of enteric neurons was investigated. Segments of guinea-pig ileum were incubated in vitro in the presence or absence of PGE2, and whole mounts of the myenteric and submucosal plexus were prepared for immunocytochemical localization of Fos, VIP and NPY. Control tissues exhibited a low number of Fos-immunoreactive (Fos-IR) neurons (7 +/- 2% of total). Incubation of the tissues with 10-1000 nM PGE2 for 30 min caused a concentration-dependent increase in Fos-IR submucosal neurons (maximum at 100 nM; 39 +/- 6%), which was not inhibited by TTX. PGE2 did not evoke an increase in Fos-IR myenteric neurons. In double labeling experiments, Fos colocalized exclusively with VIP in the submucosal plexus, and not with NPY. Exposure of stripped segments of guinea pig ileum in Ussing chambers to 100 nM PGE2 evoked an increase in short circuit current (20 +/- 7 microA/cm2), of which the initial rapid phase could be abolished by TTX, and not by atropine and hexamethonium. It is concluded that PGE2 can activate VIP non-cholinergic secretomotor neurons.     

Synaptic vesicle morphology and recycling are altered in myenteric neurons of mice lacking dystrophin (mdx mice)

Several dystrophin isoforms are known. The full-length isoform is present in striated and smooth muscles and neurons and its lack causes Duchenne Muscular Dystrophy, a progressive myopathy accompanied by mild cognitive deficits and gastrointestinal dismotility. An ultrastructural study was undertaken in the colon of mice lacking full-length dystrophin and maintaining shorter isoforms (mdx mice) to ascertain whether myenteric neurons have an altered morphology. Results showed a significant increase in the size of synaptic vesicle and in the number of recycling vesicles. An enlargement of endoplasmic reticulum cisternae in a subpopulation of neurons was also seen. Immunohistochemistry confirmed that the shorter isoforms were expressed in mdx mice myenteric neurons. These findings indicate the presence of a neuropathy at the myenteric plexus which might justify the defective neuronal control of gastrointestinal motility reported for these animals and which might be correlated with full-length dystrophin loss, since the shorter isoforms are present.     

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 microM 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.     

Hepatitis C viral infection in thalassemic children: clinical and molecular studies

The cells that express Steel factor (SLF) in the gastrointestinal (GI) tract were studied using SLF-lacZ transgenic mice. Expression, detected by beta-galactosidase histochemistry, was evident in cells between the circular and longitudinal muscle layers in the GI tract. Double staining with antibodies specific for the neural markers, PGP 9.5, MAP2 and c-Ret, showed that SLF-lacZ positive cells were enteric neurons. Enteroglia did not express SLF-lacZ. The distribution of expressing cells was complimentary to the expression of c-Kit in myenteric interstitial cells.     

c-kit-dependent development of interstitial cells and electrical activity in the murine gastrointestinal tract

In vivo injection of a neutralizing, monoclonal antibody (ACK2) to the receptor tyrosine kinase (c-kit) disrupts the normal motility patterns of the mouse small intestine. Immunohistochemical studies showed that cells expressing c-kit-like immunoreactivity (c-kit-LI) decreased in numbers in response to ACK2, but the identity of these cells is unknown. We investigated the identity and development of the cells that express c-kit-LI in the mouse small intestine and colon. Cells in the region of the myenteric plexus and deep muscular plexus of the small intestine and in the subserosa, in the myenteric plexus region, within the circular and longitudinal muscle layers, and along the submucosal surface of the circular muscle in the colon were labeled with ACK2. The distribution of cells that express c-kit-LI was the same as that of interstitial cells (ICs). In whole-mount preparations cells with c-kit-LI were interconnected, forming a network similar to the network formed by cells that stained with methylene blue, which has been used as a marker for ICs in the mouse gastrointestinal tract. Immunocytochemistry verified that ICs were labeled with ACK2. Multiple injections of animals with ACK2 between days 0 and 8 post partum (pp) caused a dramatic reduction in the number of ICs compared to control animals. From an ultrastructural point of view, the proliferation and development appeared to be suppressed in some classes of ICs, while others displayed an altered course of development. Functional studies showed that the decrease in ICs was accompanied by a loss of electrical rhythmicity in the small intestine and reduced neural responses in the small bowel and colon. Morphological experiments showed that c-kit-positive cells are ICs, and physiological evidence reinforced the concept that ICs are involved in generation of rhythmicity and translation of neural inputs in gastrointestinal smooth muscles. Controlling the development of ICs provides a powerful new tool for the investigation of the physiological role of these cells.     

The sacral neural crest contributes neurons and glia to the post-umbilical gut: spatiotemporal analysis of the development of the enteric nervous system

The majority of the enteric nervous system is derived from vagal neural crest cells (NCC), which migrate to the developing gut, proliferate, form plexuses and differentiate into neurons and glia. However, for some time, controversy has existed as to whether cells from the sacral region of the neural crest also contribute to the enteric nervous system. The aim of this study was to investigate the spatiotemporal migration of vagal and sacral NCC within the developing gut and to determine whether the sacral neural crest contributes neurons and glia to the ENS. We utilised quail-chick chimeric grafting in conjunction with antibody labelling to identify graft-derived cells, neurons and glia. We found that vagal NCC migrated ventrally within the embryo and accumulated in the caudal branchial arches before entering the pharyngeal region and colonising the entire length of the gut in a proximodistal direction. During migration, vagal crest cells followed different pathways depending on the region of the gut being colonised. In the pre-umbilical intestine, NCC were evenly distributed throughout the splanchnopleural mesenchyme while, in the post-umbilical intestine, they occurred adjacent to the serosal epithelium. Behind this migration front, NCC became organised into the presumptive Auerbach's and Meissner's plexuses situated on either side of the developing circular muscle layer. The colorectum was found to be colonised in a complex manner. Vagal NCC initially migrated within the submucosa, internal to the circular muscle layer, before migrating outwards, adjacent to blood vessels, towards the myenteric plexus region. In contrast, sacral NCC, which also formed the entire nerve of Remak, were primarily located in the presumptive myenteric plexus region and subsequently migrated inwards towards the submucosal ganglia. Although present throughout the post-umbilical gut, sacral NCC were most numerous in the distal colorectum where they constituted up to 17% of enteric neurons, as identified by double antibody labelling using the quail-cell-specific marker, QCPN and the neuron-specific marker, ANNA-1. Sacral NCC were also immunopositive for the glial-specific antibody, GFAP, thus demonstrating that this region of the neural crest contributes neurons and glia to the enteric nervous system.