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.     

Trigeminal nuclear complex of the ferret: anatomical and immunohistochemical studies

In order to establish the ferret as an animal model for studies of trigeminal pain, we describe the cytoarchitecture and neurochemistry of the trigeminal nuclear complex in the ferret and compare them to those of the cat and rat. The complex was divided as previously described, but the ferret differed in the extent of the nuclear boundaries. The neuroanatomical istribution of substance P-, calcitonin gene-related peptide-, galanin-, enkephalin-, serotonin-, somatostatin-, neuropeptide Y-, and neurotensin-immunoreactivity was determined throughout the rostrocaudal extent of the complex. In subnucleus caudalis, substance P-, calcitonin gene-related peptide-, enkephalin-, serotonin-, somatostatin-, neuropeptide Y-, and galanin-immunoreactivity was densest in laminae I and II. In subnucleus interpolaris, immunoreactivity for all the above neurochemicals was most dense along the lateral border and the ventral third of the caudal part of the subnucleus. Enkephalin-immunoreactive cell bodies were present in subnucleus caudalis and interpolaris. In subnucleus oralis, labelling for substance P, calcitonin gene-related peptide, galanin, enkephalin, and serotonin was most prominent in the dorsomedial part of the subnucleus. Somatostatin-immunoreactive cell bodies were distributed throughout the spinal nucleus. Labelling of serotonin, substance P, calcitonin gene-related peptide, galanin, enkephalin, and somatostatin was present in the main sensory nucleus. The motor nucleus contained fibers immunoreactive for substance P, enkephalin, serotonin and neuropeptide Y, and cell bodies immunoreactive for calcitonin gene-related peptide. The majority of neurotensin-immunoreactivity was found at the level of subnucleus caudalis, where it was densest in the trigeminal extension of the lateral cervical nucleus. The distribution of peptides in this species throughout the spinal nucleus is consistent with the notion that all the subnuclei may be involved in the processing of nociceptive inputs.     

Contractile response of isolated human hepatic arteries to alpha-adrenoceptor agonists is not impaired in patients with cirrhosis

The endogenous opioid peptide dynorphin has been shown by immunochemical studies to be widely distributed in the gastrointestinal tract. The aim of this study was to determine basal levels of preprodynorphin (ppDyn) mRNA in different regions of the gastrointestinal tract of the guinea pig. A modified sensitive and specific solution hybridization RNase protection assay was used to quantitate ppDyn mRNA, with confirmation by gel analysis of the RNase protected hybrids and PCR amplified cDNA. This method combines high sensitivity and sufficient throughput to analyze large number of samples in a single assay. Low but measurable amounts of ppDyn mRNA were detected in fundus, duodenum, jejunum, ileum, cecum, and rectum. The rectum contained significantly more ppDyn mRNA than the stomach, small bowel, and cecum. The muscularis/myenteric plexus layer of both ileum and rectum contained a higher concentration of ppDyn mRNA per microg total RNA compared to the mucosa/submucosa/submucosal plexus. However, a greater absolute amount of ppDyn mRNA (80-85%) localized to the mucosal layer. The greater absolute amount of ppDyn mRNA in the mucosal layer may indicate the presence of dynorphin in the endocrine cells of the mucosa.     

Serotonin accumulation in the guinea-pig myenteric plexus: ion dependence, structure-activity relationship and the effect of drugs

The specific accumulation of serotonin in neurons of the guinea-pig myenteric plexus was examined. Nonadrenergic neurons have been shown to be responsible for this accumulation. Serotonin accumulation, known to be sodium-dependent, was found to be inhibited by elevating the external potassium concentration. Accumulation was inversely related to the concentration of potassium and was also decreased in the presence of 0 mM Ca++ or 12 mM Ca++. The affinity of analogous molecules for the myenteric plexus was greatly reduced in compounds which had no alkyl amino side chain, in which the amino group was methylated or which had no 5-hydroxyl group. Most analogs competitively inhibited serotonin accumulation and 6-hydroxytryptamine was demonstrated by histofluorescence to be taken up into the myenteric plexus after chemical sympathectomy with 6-hydroxydopamine. Serotonin accumulation was also inhibited by tricyclic antidepressants and amphetamines. The inhibition of serotonin accumulation by these compounds differed from their inhibition of accumulation of norepinephrine. As in the central nervous system, chlorimipramine was the most potent tricyclic antidepressant against accumulation of serotonin while desmethylimipramine was the most potent inhibitor of the accumulation of norepinephrine. Amphetamines were more effective inhibitors of serotonin accumulation than were tricyclic antidepressants, but all of these drugs were more effective against the accumulation of norepinephrine than serotonin. This study confirms the existence of a unique population of axons int he mammalian myenteric plexus which are distinguishable by their characteristic accumulation of serotonin and have not been found elsewhere in the peripheral nervous system.     

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.     

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.     

Colocalization of substance P or enkephalin in serotonergic neuronal afferents to the hypoglossal nucleus in the rat

The serotonergic innervation of the hypoglossal nucleus originates from the caudal raphe nuclei. Non-serotonergic neurons in the caudal raphe nuclei also project to the hypoglossal nucleus. We employed a triple-fluorescence technique to determine whether the substance P- or the enkephalin-containing neurons in the caudal raphe nuclei that projected to the hypoglossal nucleus also contained serotonin. Rhodamine latex microspheres were injected into the hypoglossal nucleus, and then serotonin and peptide dual-immunofluorescence was performed to colocalize perikarya containing serotonin, substance P, and rhodamine microspheres; or perikarya containing serotonin, enkephalin, and rhodamine microspheres. Our results demonstrate that most substance P-containing neuronal afferents to the hypoglossal nucleus colocalize serotonin. In contrast, few enkephalin-containing neuronal afferents to the hypoglossal nucleus also contain serotonin. These data suggest that substance P projections to the hypoglossal nucleus are a subset of serotonergic projections and that limited overlap exists between the populations of enkephalinergic and serotonergic neuronal afferents to the hypoglossal nucleus. Either substance P- or enkephalin-containing somata account for a very small proportion of non-serotonergic caudal raphe projections to the hypoglossal nucleus. Finally, these data demonstrate the medial tegmental field origins of the substance P projections and the enkephalin projections to the hypoglossal nucleus.     

The art of nursing

The specific motility patterns of the forestomach of ruminants, composed of three structurally distinct compartments (rumen, reticulum, omasum), require an elaborate intramural innervation. To demonstrate the complex structure of the enteric nervous system (ENS), whole mount preparations obtained from different sites of the bovine forestomach were submitted to immunohistochemical procedures in which neuronal (protein gene product 9.5, neurofilament 200) and glial (protein S-100, glial fibrillary acid protein) markers were applied. Immunohistochemistry performed on whole mounts allowed a detailed two-dimensional assessment of the architecture of the intramural nerve networks. Generally, the myenteric and submucosal plexus layers were composed of ganglia and interconnecting nerve fiber strands, whereas the mucosal plexus consisted of an aganglionated nerve network. However, the texture of the ENS showed considerable regional differences concerning the ganglionic size, shape and density and the arrangement of nerve fiber strands. The myenteric plexus of the ruminal wall, showing a low ganglionic density and wide polygonal meshes, contrasted with the nerve network within the ruminal pillar which consisted of ropeladder-like nerve fiber strands and parallel orientated ganglia. The highest ganglionic density was observed at the reticular groove, the most prominent ganglia were found within the omasal wall. Branches of the vagal nerve frequently ramified within the myenteric plexus layers. The submucosal plexus of the rumen was divided into an external and internal layer; the reticular submucosal plexus followed the cristae and cellulae reticuli, the omasal submucosal (sublaminar) plexus showed intra- and parafascicular ganglia apart from ganglia located at the junctions of the nerve network. The mucosal plexus of the rumen consisted of thin nerve fascicles ramifying between the ruminal papillae, and reticular mucosal nerve fibers passed throughout the base of the cellulae reticuli. The highly specialised nerve network of the intralaminar omasal plexus showed radial and transverse trajectories reflecting the spatial arrangement of the intralaminar musculature. The demonstrated structural complexity of the ENS reflects the functional complexity of the ruminant forestomach and indicates the relatively high degree of autonomy in coordinating the different motility patterns required for the processing of the ingesta.     

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.     

Peptone stimulates CCK-releasing peptide secretion by activating intestinal submucosal cholinergic neurons

In this study we tested the hypothesis that peptone in the intestine stimulates the secretion of the CCK-releasing peptide (CCK-RP) which mediates CCK secretion, and examined the enteric neural circuitry responsible for CCK-RP secretion. We used a "donor-recipient" rat intestinal perfusion model to quantify the CCK-RP secreted in response to nutrient stimulation. Infusion of concentrated intestinal perfusate collected from donor rat perfused with 5% peptone caused a 62 +/- 10% increase in protein secretion and an elevation of plasma CCK levels to 6.9 +/- 1.8 pM in the recipient rat. The stimulatory effect of the intestinal washings was abolished when the donor rats were pretreated with atropine or hexamethonium but not with guanethidine or vagotomy. Mucosal application of lidocaine but not serosal application of benzalkonium chloride which ablates the myenteric neurons in the donor rats also abolished the stimulatory action of the intestinal washings. Furthermore, treatment of the donor rats with a 5HT3 antagonist and a substance P antagonist also prevented the secretion of CCK-RP. These observations suggest that peptone in the duodenum stimulates serotonin release which activates the sensory substance P neurons in the submucous plexus. Signals are then transmitted to cholinergic interneurons and to epithelial CCK-RP containing cells via cholinergic secretomotor neurons. This enteric neural circuitry which is responsible for the secretion of CCK-RP may in turn play an important role in the postprandial release of CCK.     

Colocalization of nitric oxide synthase and some neurotransmitters in the intramural ganglia of the guinea pig urinary bladder

The distribution of nitrergic neurons was investigated by using nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemistry and nitric oxide synthase (NOS) immunohistochemistry in wholemount preparations of the urinary bladder in guinea pigs. Both NADPH-d+ and NOS+ neurons were located predominantly in the bladder base. Double staining showed that 70.9% of the NADPH-d+ neurons coexpressed NOS. Acetylcholinesterase histochemistry revealed that a majority of the intramural neurons were reactive, and about half of them (51.4%) were double labelled for NOS. Tyrosine hydroxylase-positive neurons were also distributed mainly in the bladder base but in a neuronal population that was separate from the preponderant NADPH-d+ neurons. Vasoactive intestinal polypeptide immunoreactivity was also detected in the some of intramural ganglion cells, in which 21.3% of them coexpressed NADPH-d. Calcitonin gene-related peptide and substance P immunoreactivities were confined to nerve fibers, often in close association with NADPH-d+ cells or extended along the blood vessels. These results have demonstrated the colocalization of NADPH-d and NOS in the majority of intramural ganglion cells. Many of the nitrergic neurons are apparently cholinergic, indicating that they are parasympathetic postganglionic neurons, and this underscores NO as the major neuromodulator in the parasympathetic nerves in the bladder walls. The localization of vasoactive intestinal polypeptide in nitrergic neurons suggests that the peptide may complement NO for regulation of micturition reflex. The close relationship of NADPH-d-reactive intramural neurons with calcitonin gene-related peptide and substance P fibers, most probably derived from dorsal root ganglion cells, suggests that NO released from the local neurons may exert its influence on the sensory neural pathways in the urinary bladder.     

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.     

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.     

Morphometry and acetylcholinesterase activity of the myenteric plexus of the wild mouse Calomys callosus

The myenteric plexus of the digestive tract of the wild mouse Calomys callosus was examined using a histochemical method that selectively stains nerve cells, and the acetylcholinesterase (AChE) histochemical technique in whole-mount preparations. Neuronal density was 1,500 +/- 116 neurons/cm2 (mean +/- SEM) in the esophagus, 8,900 +/- 1,518 in the stomach, 9,000 +/- 711 in the jejunum and 13,100 +/- 2,089 in the colon. The difference in neuronal density between the esophagus and other regions was statistically significant. The neuron profile area ranged from 45 to 1,100 microns2. The difference in nerve cell size between the jejunum and other regions was statistically significant. AChE-positive nerve fibers were distributed within the myenteric plexus which is formed by a primary meshwork of large nerve bundles and a secondary meshwork of finer nerve bundles. Most of the nerve cells displayed AChE activity in the cytoplasm of different reaction intensities. These results are important in order to understand the changes occurring in the myenteric plexus in experimental Chagas' disease.     

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.     

Capsaicin-resistant vagal afferent fibers in the rat gastrointestinal tract: anatomical identification and functional integrity

The presence and distribution of vagal fibers and terminals throughout esophagus and gastrointestinal tract that could be anterogradely labeled by nodose ganglion tracer injections was quantitatively assessed in capsaicin- and vehicle-pretreated adult rats, in order to identify the capsaicin-resistant population. Up to 90% of the intraganglionic laminar endings (IGLEs), in the myenteric plexus of the esophagus, and 70-90% in the stomach, as well as 57% of the intramuscular endings or arrays (IMAs) in the fundic stomach survived the capsaicin treatment, while in the upper small intestine only few and in the lower small intestine, the cecum and colon, virtually no IGLEs survived capsaicin treatment. Intramucosal terminals were not assessed. Furthermore, gastric balloon distension-induced c-Fos expression in the dorsal vagal complex was not significantly decreased in capsaicin-treated rats. It is concluded that among primary vagal afferents there is a capsaicin-resistant population that primarily innervates the esophagus and upper gastrointestinal tract, and a capsaicin-sensitive population that innervates mainly the lower tract. At least vagal gastric tension-sensitive afferents also seems to be functionally intact in that they may be capable of synaptically activating second-order neurons in the brainstem.     

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.     

The presence of aromatic L-amino acid decarboxylase in certain intestinal nerve cells

The presence of aromatic 1-amino acid decarboxylase (AADC) in nerve cell bodies of the intrinsic plexuses of the guinea-pig small intestine was demonstrated by incubating segments of intestine with 1-dopa in the presence of an inhibitor of monoamine oxidase, pargyline. After such incubation, some nerve cell bodies gave a fluorescence histochemical reaction indicative of the presence of a decarboxylated product of 1-dopa, probably dopamine. No fluorescence reaction occurred in the unincubated control or if the inhibitor of AADC, RO 4-4602, was included in the incubation mixture. The AADC-containing cell bodies apparently do not take up and store dopamine, because no fluorescence could be detected after incubation with dopamine and a monoamine oxidase inhibitor. The AADC-containing cells were found in about half of the ganglia of the submucous plexus of the guinea-pig small intestine, but were considerably less frequent in the myenteric plexus. They were also found in the other areas examined in this study, that is, in both enteric plexuses of the guinea-pig distal colon and of the small intestines of rabbits and rats.     

Arthroscopic-assisted allograft anterior cruciate ligament reconstruction in patients with symptomatic arthrosis

BACKGROUND: Antral motility and the hormone cholecystokinin (CCK) are major determinants of the rate of gastric emptying. The relation between CCK and antral neurons in regulating gastric emptying is uncertain. Benzalkonium chloride (BAC) causes selective lesions in gut myenteric neurons after serosal application. AIM: To develop a model of antral denervation using BAC to enable the study of the relation between CCK and antral neurons in regulating gastric emptying. METHODS: BAC, vehicle or the afferent neurotoxin capsaicin were applied to the serosal surface of the rat antrum or corpus; neurochemical markers of intrinsic and afferent neurons were detected by using immunohistochemistry and radioimmunoassay. Gastric retention of solids was determined after fasting, and emptying of liquids was measured in rats with gastric fistulae. RESULTS: In BAC treated rats radioimmunoassay of tissue extracts revealed a dose related specific loss of gastrin releasing peptide, substance P, and vasoactive intestinal polypeptide immunoreactivities from the treated region, and immunohistochemistry revealed loss of the neuronal marker PGP 9.5 and the afferent neuropeptide calcitonin gene related peptide (CGRP). Adjacent untreated regions were unaffected by BAC, with the exception that CGRP was depleted in both corpus and antrum after antral treatment. After antral BAC treatment fasted rats retained solids for over 48 hours. Moreover, in antrally denervated rats with gastric fistulae, the emptying of saline, acid and peptone was delayed substantially. The CCK dependent inhibition of gastric emptying of peptone was preserved after antral treatment with BAC. CONCLUSIONS: Serosal BAC causes lesions in the innervation of the treated region of the stomach. The innervation of the antrum is essential for normal emptying of both liquids and solids, but the inhibition of gastric emptying produced by CCK is not dependent on antral neurons.     

Differentiation of enteric plexuses and interstitial cells of Cajal in the rat gut during pre- and postnatal life

The stomach, small and large intestine of fetuses at term, of unfed newborns, of suckling, weaning and of adult rats were studied by a combined light (LM) and electron microscope (EM) examination. Neuron-specific enolase was used as a neuronal marker under LM. Zinc-iodide-osmium (ZIO) impregnation was used for a selective staining of neurons and interstitial cells of Cajal under both LM and EM. A routine EM procedure made it possible to identify the nerve elements and ICC and to evaluate their degree of differentiation. The differentiation of enteric plexuses and ICC was poor at birth and was accomplished during the weaning period. The myenteric plexus differentiation preceded the submucous plexus differentiation; in particular, under both LM and EM myenteric neurons were already recognizable in the fetus, while the submucous neurons by day 0 under EM and by day 7 under LM. The ICC were poorly differentiated at birth and acquired the adult morphology during the suckling period. Nerve endings contacting ICC were differentiated before ICC differentiation. The ZIO uptake by both nerve elements and ICC correlated with the establishment of their differentiated features. In conclusion, the present findings confirmed that differentiation of ICC and enteric plexuses is microenvironment dependent, since their differentiative steps are interrelated and correlated with diet changes. ZIO impregnation under EM enabled a distinction to be made between a "morphological' and a "functional' differentiation, and revealed that the former is achieved during the suckling period and the latter by the weaning period. It can be suggested that during the postnatal developmental stages ICC and neuronal functions might be different from those in adulthood.