Effect of age on the enteric nervous system of the human colon.

The effect of age on the anatomy and function of the human colon is incompletely understood. The prevalence of disorders in adults such as constipation increase with age but it is unclear if this is due to confounding factors or age-related structural defects. The aim of this study was to determine number and subtypes of enteric neurons and neuronal volumes in the human colon of different ages. Normal colon (descending and sigmoid) from 16 patients (nine male) was studied; ages 33-99. Antibodies to HuC/D, choline acetyltransferase (ChAT), neuronal nitric oxide synthase (nNOS), and protein gene product 9.5 were used. Effect of age was determined by testing for linear trends using regression analysis. In the myenteric plexus, number of Hu-positive neurons declined with age (slope = -1.3 neurons/mm/10 years, P = 0.03). The number of ChAT-positive neurons also declined with age (slope = -1.1 neurons/mm/10 years of age, P = 0.02). The number of nNOS-positive neurons did not decline with age. As a result, the ratio of nNOS to Hu increased (slope = 0.03 per 10 years of age, P = 0.01). In the submucosal plexus, the number of neurons did not decline with age (slope = -0.3 neurons/mm/10 years, P = 0.09). Volume of nerve fibres in the circular muscle and volume of neuronal structures in the myenteric plexus did not change with age. In conclusion, the number of neurons in the human colon declines with age with sparing of nNOS-positive neurons. This change was not accompanied by changes in total volume of neuronal structures suggesting compensatory changes in the remaining neurons.

Apoptotic cell death of human interstitial cells of Cajal.

Interstitial cells of Cajal (ICC) are specialized mesenchyme-derived cells that regulate contractility and excitability of many smooth muscles with loss of ICC seen in a variety of gut motility disorders. Maintenance of ICC numbers is tightly regulated, with several factors known to regulate proliferation. In contrast, the fate of ICC is not established. The aim of this study was to investigate whether apoptosis plays a role in the regulation of ICC numbers in the normal colon. ICC were identified by immunolabelling for the c-Kit receptor tyrosine kinase and by electron microscopy. Apoptosis was detected in colon tissue by immunolabelling for activated caspase-3, terminal dUTP nucleotide end labelling and by ultrastructural changes in the cells. Apoptotic ICC were identified and counted in double-labelled tissue sections. They were identified in all layers of the colonic muscle. In the muscularis propria 1.5 +/- 0.2% of ICC were positive for activated caspase-3 and in the circular muscle layer 2.1 +/- 0.9% of ICC were positive for TUNEL. Apoptotic ICC were identified by electron microscopy. Apoptotic cell death is a continuing process in ICC. The level of apoptosis in ICC in healthy colon indicates that these cells must be continually regenerated to maintain intact networks.

Distribution of interstitial cells of Cajal and nitrergic neurons in normal and diabetic human appendix.

The objective of this study was to determine the distribution of enteric nerves and interstitial cells of Cajal (ICC) in the normal human appendix and in type 1 diabetes. Appendixes were collected from patients with type 1 diabetes and from non-diabetic controls. Volumes of nerves and ICC were determined using 3-D reconstruction and neuronal nitric oxide synthase (nNOS) expressing neurons were counted. Enteric ganglia were found in the myenteric plexus region and within the longitudinal muscle. ICC were found throughout the muscle layers. In diabetes, c-Kit positive ICC volumes were significantly reduced as were nNOS expressing neurons. In conclusion, we describe the distribution of ICC and enteric nerves in health and in diabetes. The data also suggest that the human appendix, a readily available source of human tissue, may be useful model for the study of motility disorders.

Kit/stem cell factor receptor-induced phosphatidylinositol 3'-kinase signalling is not required for normal development and function of interstitial cells of Cajal in mouse gastrointestinal tract.

Signalling mediated by the receptor tyrosine kinase c-Kit is required for normal development of interstitial cells of Cajal (ICC). c-Kit activates several signalling pathways, including the phosphatidylinositol 3'-kinase (PI3'-kinase) pathway. The signals required for ICC development and maintenance are not well understood. Studies indicate a role for PI3'-kinase. We studied ICC function and morphology in mice homozygous for the tyrosine 719 to phenylalanine c-Kit mutation, which disrupts all PI3'-kinase binding to c-Kit. Functionally, the electrical slow waves in the jejunum and inhibitory junction potentials were normal in adult mutants. Morphologically, the distribution of ICC was not altered in mutants. There was no difference in the density of ICC in the jejunum of adults or newborns from quantitative analysis of c-Kit immunoreactivity. The number of ICC obtained in culture was the same using mutants or wild-type littermates. The density and organization of nerves in the jejunum of mutants was not affected. Deletion of c-Kit-induced PI3'-kinase signalling does not affect the function or development of ICC in the mouse. This is an important and counterintuitive result, given the role of PI3'-kinase signalling downstream of c-Kit and the role of both c-Kit and PI3'-kinase individually in ICC development.

Morphological characteristics and immunohistochemical detection of nicotinic acetylcholine receptors on intestinofugal afferent neurones in guinea-pig colon.

Intestinofugal afferent neurones (IFANs) provide excitatory synaptic input to abdominal prevertebral ganglion neurones. Input is greatly reduced during blockade of nicotinic acetylcholine receptors (nAChRs) in the wall of the colon, suggesting two projection pathways: a direct pathway without synaptic interruption and an indirect pathway interrupted by at least one nicotinic cholinergic synapse. This study aimed to characterize the morphology of IFANs and examine the distribution of nAChRs on them. We identified IFANs in guinea-pig colon by retrograde labelling with fluorescent tracer DiI placed either on the lumbar colonic nerves in vitro or inferior mesenteric ganglion in vivo. Confocal laser scanning microscopy and computerized image-processing software were used for 3D image reconstruction. Approximately 70% of identified IFANs had Dogiel type I-like morphology, the remainder were Dogiel type II-like. In vivo labelled IFANs were injected with Lucifer Yellow and immunostained for nAChRs using monoclonal antibody MAb35. Approximately 3% of total plasma membrane surface of IFANs with Dogiel type I morphology had MAb35-IR. In contrast, <1% of membrane surface of IFANs with Dogiel type II morphology had MAb35-IR. The finding that IFANs displayed immunostaining for nAChRs suggests the presence of putative nicotinic synapses.

The three-dimensional structure of neurons in the guinea pig inferior mesenteric and pelvic hypogastric ganglia.

The three-dimensional (3-D) morphology of sympathetic inferior mesenteric ganglion (IMG) neurons and sympathetic-parasympathetic pelvic hypogastric ganglion (PHG) neurons was studied using confocal laser scanning microscopy. Cell bodies of IMG neurons were disc-shaped and were arranged orderly in layers. The dendritic arbor of individual neurons was confined to a plane with a thickness that did not exceed the thickness of the parent cell body. The actual dendritic surface area (71,400 micron 2) and volume (81,500 micron 3) of the IMG neurons were up to 100-fold larger than previously reported for similar sympathetic neurons using data of 2-D measurements and estimations of the third dimension. PHG neurons had a much smaller dendritic surface area (4100 micron 2) and volume (2400 micron 3) compared to IMG neurons. The ratio dendritic/somal surface area for individual IMG and PHG neurons ranged from 5:1 to 14:1 and from 0.1:1 to 6:1, respectively. The total dendritic path-length was 8-42 times greater for IMG than for PHG neurons. Neurons in the IMG were either stellate with radiating dendrites or bipolar-shaped with dendrites emerging from the two poles of the cell body. Neurons in the PHG were of two morphological types. One type (nearly 2/3 of all the imaged PHG neurons) had two to seven relatively long dendrites and an axon; the other type had only one to three short unbranched dendrites and an axon. The spatial organization of neurons within the ganglia and the structural features of individual neurons are likely to have important implications regarding connectivity patterns between neurons within the ganglion as well as on how information is processed by the ganglion.

Leptin receptor immunoreactivity in sympathetic prevertebral ganglion neurons of mouse and rat.

Prevertebral ganglia, comprising the inferior mesenteric, superior mesenteric and celiac ganglia, contain the cell bodies of sympathetic neurons that regulate gastrointestinal motility, visceral blood flow, secretion and absorption. In the present study, we report the presence of leptin receptor-like immunoreactivity in the superior mesenteric and celiac ganglia of mice and rats. Ganglion neurons were immunopositive for leptin receptor. Confocal microscopy and three-dimensional (3-D) reconstruction of ganglion neurons filled with Lucifer yellow and then immunostained for leptin receptor showed that leptin receptor immunoreactivity was intracellular and that it was present on structures encircling the nucleus. These results raise the possibility that leptin may affect gastrointestinal function by acting on leptin receptors located in prevertebral ganglion neurons.

The three-dimensional structure of myenteric neurons in the guinea-pig ileum.

Myenteric neurons of the guinea-pig ileum were intracellularly filled with the fluorescent dye Lucifer Yellow, optically sectioned with a confocal microscope and volume reconstructed to recreate 3-D images of the cells. The resulting images provide information not evident from regular microscopy. The somata varied in cross-section from flat-oval to nearly circular, and their surface membranes were marked by invaginations and protrusions significantly increasing the surface area of the somatic membrane. The neurons could be divided into four morphological classes: Dogiel type I, Dogiel type II, filamentous, and intermediate. There was no clear correlation between cell class and the shape of the soma in cross-section. The dendritic processes of all the neurons studied extended in an orad-caudad or circumferential direction of the bowel wall. When the filled neurons were viewed edge-on, the spatial arrangement of the processes was confined to a plane that had a thickness less than the thickness of the parent soma. The broad, short dendrites of Dogiel type I neurons were oval or nearly circular in cross-section. Directly measured quantitative data were obtained for the volume and surface area of the somata and visible processes. The structural details reported herein are likely to have important implications regarding the functional properties of individual enteric ganglion neurons and circuits of enteric ganglion neurons.

Heme oxygenase 2 is present in interstitial cell networks of the mouse small intestine.

BACKGROUND & AIMS: The interstitial cell (IC) network may be of fundamental importance in regulating gastrointestinal motility. Intestinal smooth muscle cells are depolarized in the absence of ICs, and there are no spontaneous slow waves. The messenger molecules between IC network and smooth muscle are unknown. Exogenous administration of CO relaxes the opossum internal anal sphincter and the guinea pig ileum, and it modulates potassium current and membrane potential of circular smooth muscle cells of the human jejunum. The aim of this study was to determine whether heme oxygenase (HO)-1 and HO-2, enzymes that catalyze the production of CO, are present in the IC network of the mouse small intestine. METHODS: Antibodies specific for c-Kit, HO-1, and HO-2 were used for immunohistochemistry. Confocal images were obtained and were volume rendered, and the images were converted into three-dimensional images. RESULTS: HO-2-like but not HO-1-like immunoreactivity was found in IC networks associated with the myenteric plexus and the deep muscular plexus. CONCLUSIONS: HO-2 but not HO-1 is present in the IC cell network of the mouse small intestine. The enzymatic activity of HO-2 will result in the endogenous production of CO in IC networks of the mouse small intestine.

Light, electron, and confocal microscopic study of the mouse superior mesenteric ganglion.

The superior mesenteric ganglion (S.m.g.), a sympathetic prevertebral ganglion, is an integrating center for gastrointestinal reflexes. Many details of its structure are still lacking. In the present study, mouse S.m.g. neurons were studied by light, electron, and confocal microscopy. Neurons had an average of 5-6 primary dendrites. Total dendritic length averaged 963 microns. Confocal microscopy and three-dimensional reconstructed images revealed cell body surface features, precise location where axons and dendrites emerged from it, cell body size, and extent of dendritic projection in three axes. Cell body diameter and dendritic projections were less in the dorsoventral than in the rostrocaudal or mediolateral axes. Cell body surface area and volume averaged 4,271 microns 2 and 4,908 microns 3, respectively. Dendritic surface areas and volumes were 5-6 times larger. Two main neuron types (projecting caudally or rostrally) were distinguished. The former were found throughout the S.m.g., whereas the latter were found only in the cephalad region, comprising about 40% of neurons found there. Rostrally projecting neurons had fewer primary dendrites, fewer total dendritic branches, and shorter total dendritic length than caudally projecting neurons. There were regional differences in percentage of neurons responding to electrical stimulation of left or right hypogastric, lumbar colonic, or left splanchnic nerves but not in nerve fibers connecting the S.m.g. and celiac ganglion. A greater percentage of caudally than rostrally projecting cephalad neurons responded to stimulation of any nerve trunk. These results indicate that the mouse S.m.g. contains at least two distinct types of neurons that differ in their morphology and their source of preganglionic synaptic input.

Bovine gallbladder muscularis: source of a myogenic receptor for cholecystokinin.

Despite being a classic target for the gastrointestinal peptide hormone, cholecystokinin (CCK), the gallbladder CCK receptor is not well characterized. Pharmacological studies of small species suggest that CCK action can be mediated by direct myogenic or by both myogenic and neurogenic receptors. To prepare for the biochemical characterization of a gallbladder CCK receptor and to define the subtype of the receptor being studied, we have performed autoradiographic localization and pharmacological characterization of CCK receptors on bovine gallbladder. Autoradiography demonstrated high-affinity specific CCK-binding sites only on the muscularis. CCK-8 stimulated tonic contraction of longitudinal strips of gallbladder muscularis in a concentration-dependent manner, with an ED50 of 0.2 nM. Antagonism at the cholinergic receptor with 1 microM atropine or axonal transmission with 1 microM tetrodotoxin did not modify CCK-induced contraction, supporting a direct myogenic effect of this hormone. Optimal electrical field stimulation (10 V, 10 Hz, 500 microseconds) to elicit a neuronal response resulted in muscle strip relaxation, which was abolished with adrenergic blockade (1 microM phentolamine, 1 microM propranolol). Although acetylcholine administration stimulated contraction, electrical field stimulation did not, even in the presence of phentolamine, propranolol, and/or CCK. Thus, in bovine gallbladder muscularis, there is evidence for a functional CCK receptor only on smooth muscle cells. Demonstration of a single, high-affinity specific CCK-binding site on an enriched plasma membrane preparation of bovine gallbladder muscularis is consistent with this representing a myogenic CCK receptor.

Intracellular electrical activity of muscularis mucosae of the dog stomach.

Mechanical and intracellular electrical activity was recorded simultaneously in vitro from smooth muscle of the muscularis mucosae of the canine antrum. The intracellularly recorded membrane potential averaged -51 +/- 1.4 mV (mean +/- SE). Spontaneous electrical activity consisted of spike-shaped potentials that were 20-40 mV in amplitude. The rate of rise of the spike potential was slow (less than 0.2 V/s) and the half-time duration was long (0.5-5.0 s). Phasic contractions were often but not always coupled with spike potentials. Ion substitution studies suggested that the spike potential had a greater dependence on Na+ than on Ca2+. Field stimulation of intramural nerves hyperpolarized the membrane potential and abolished spikes or reduced their amplitude and frequency. These changes were associated with a reduction in tone and phasic contractile activity. The response to stimulation of inhibitory nerves was mimicked by epinephrine, neurotensin, and vasoactive intestinal polypeptide. The resistance to adrenergic blocking agents ruled out the possibility of norepinephrine as the transmitter. The tetrodotoxin sensitivity of the response to neurotensin suggests that neurotensin acts indirectly through the inhibitory nerves. Mimicry between the action of applied vasoactive intestinal polypeptide (VIP) and field stimulation provides support for the hypothesis that VIP may be an inhibitory neurotransmitter. These studies indicate that smooth muscle in the canine gastric muscularis mucosae generates spontaneous electrical and mechanical activity and receives a noncholinergic, nonadrenergic inhibitory innervation.

Pentagastrin potentiates nonadrenergic inhibitory neuromuscular transmission in orad stomach of the dog.

Mechanical and intracellular electrical activities were recorded separately from circular muscle of the orad corpus of the canine stomach. In normal Krebs solution, transmural electrical nerve stimulation produced excitatory and inhibitory responses. The excitatory response was blocked by atropine; the inhibitory response was unaffected by phentolamine or propranolol alone or in combination. Both responses were blocked by tetrodotoxin. In the presence of pentagastrin, the inhibitory response to electrical nerve stimulation was potentiated in a dose-dependent manner. The threshold concentration for this potentiating effect was 3 X 10(-11) M. Intracellular recordings showed that hyperpolarization of the resting membrane potential in response to inhibitory nerve stimulation was greater in the presence of pentagastrin than in its absence. These data suggest that, in addition to a direct stimulatory effect on the muscle, pentagastrin also potentiates the effect of ongoing activity of intramural inhibitory nerves. This latter effect is only apparent when the inhibitory neural network is active. Thus, the in vivo effect of pentagastrin on motor activity of the corpus will depend on the degree of ongoing activity of intramural inhibitory nerves.

Vasoactive intestinal polypeptide: a putative transmitter in the canine gastric muscularis mucosa.

The nature of the inhibitory transmitter in the canine gastric muscularis mucosae was studied in vitro using superfusion techniques. The inhibitory effect of nerve stimulation (10 V, 200 mus, 10 Hz) was not altered by adrenergic, cholinergic or serotonergic antagonists. Adenosine triphosphate had no effect on spontaneous mechanical activity. Nucleotide pyrophosphatase and apamin had no effect on the response to nerve stimulation. Alpha-chymotrypsin abolished the inhibitory effect of nerve stimulation. Radioimmunoassay of the muscle indicated the presence of gastrin/cholecystokinin-substance P- and vasoactive intestinal polypeptide (VIP)-like immunoreactivity. Of the three peptides present, only VIP produced a concentration-dependent relaxation. A substance with VIP-like immunoreactivity was released during nerve-induced relaxation of the muscle, and its release was blocked by tetrodotoxin and calcium-depleted solution. The inhibitory effect of nerve stimulation was abolished by VIP antiserum. These data strongly support the hypothesis that VIP or a closely related peptide is an inhibitory neurotransmitter in the canine gastric muscularis mucosae.

Central innervation of neurones in the inferior mesenteric ganglion and of the large intestine of the cat.

1. Segmental, lumbar sympathetic outflow to neurones in the cat inferior mesenteric ganglion and to the large intestine were studied. Synaptic responses of neurones in the inferior mesenteric ganglion were recorded intracellularly, in vitro, during electrical stimulation of preganglionic fibres in the lumbar white rami. Synaptic responses consisted of excitatory post-synaptic potentials and/or action potentials.2. None of the neurones tested received synaptic input from spinal cord segment L(1). There was synaptic input from segments L(2)-L(5) of the spinal cord. The strongest synaptic input arose from spinal cord segments L(3) and L(4).3. 42% of the neurones tested received synaptic input from only one spinal cord segment. 54% of the neurones tested received convergent synaptic input from two, three or four adjacent lumbar segments.4. Electrophysiological measurements indicated that the number of preganglionic fibres in any lumbar white ramus communicans which provided synaptic input ranged from one to thirteen. Each lumbar white ramus contained, on average, five preganglionic fibres which provided synaptic input to neurones in the inferior mesenteric ganglion.5. Changes in intraluminal colonic pressure were measured in vivo during electrical stimulation of preganglionic fibres in the different lumbar white rami and lumbar ventral roots. Electrical stimulation of white rami L(3) and L(4) abolished phasic changes in intraluminal colonic pressure and reduced basal pressure to near zero. Electrical stimulation of preganglionic fibres in lumbar ventral roots L(3) and L(4) abolished phasic changes in intraluminal colonic pressure and reduced basal pressure to near zero. Stimulation of ventral roots L(1), L(2) and L(5) had little to no effect on intraluminal pressure.6. Based on the data obtained in this study, two hypotheses are proposed. First, spinal cord segments L(3), L(4) and L(5) are the primary sources of central synaptic input to neurones in the inferior mesenteric ganglion. Secondly, spinal cord segments L(3) and L(4) control colonic motility.

Innervation of the muscularis mucosa in the canine stomach and colon.

A preliminary report is presented of studies into the innervation of the canine muscularis mucosa. In vitro experiments have investigated the mechanical response to electrical stimulation and the release of gut peptides known to modulate gastrointestinal function. Exogenous substance P increased tone and amplitude of phasic contractions while neurotensin and vasoactive intestinal polypeptide (VIP) relaxed the muscularis mucosa. Immunoreactive VIP was released into the superfusate during transmural electrical stimulation of the antrum while VIP and substance P were present in superfusate from the colon. The significance of these findings has been discusses briefly.

Effects of pentagastrin, G17, and G34 on the electrical and mechanical activities of canine antral smooth muscle.

Intracellular microelectrode technique and standard organ bath technique were used to investigate the effects of pentagastrin, G17, and G34 on the electrical and mechanical activities of canine antral circular muscle. All three molecular forms increased the amplitude and duration of the plateau of the gastric action potential and the frequency of spontaneous action potentials. They also increased the amplitude and frequency of spontaneous contractions. G17 was equal to or less potent than pentagastrin in all of its actions on this tissue. G34 had an equal or greater activity than G17. The electrical studies indicate that G17 is active in this tissue in a physiological range of concentrations. The ED50 for the effect of G17 to increase the amplitude of the plateau potential is less than that for the effect of G17 on gastric secretion, indicating that this is a physiological action of gastrin. Atropine studies indicate that only part of the in vitro inotropic action of gastrin is caused by the release of acetylcholine from nerve terminals, but that the chronotropic action is attributable to a direct effect on the smooth muscle membrane.

Electrical and mechanical effects of molecular variants of CCK on antral smooth muscle.

Intracellular microelectrode and standard organ bath techniques were used to study in vitro the effects of three molecular forms of the peptide cholecystokinin on the electrical and mechanical activities of canine antral circular muscle. Three forms were studied: the carboxyl-terminal octapeptide of cholecystokinin (CCK-OP), the molecule containing 33 amino acid residues (CCK33), and the peptide termed "cholecystokinin variant" that contains 39 amino acids (CCK39). All three forms increased the force and frequency of spontaneous contractions. They also increased the frequency and the amplitude and duration of the plateau of the gastric action potential. Atropine did not block any of these effects, suggesting that the action of these peptides was largely due to a direct action on the smooth muscle. Complete dose-response curves were determined for the effect of these peptides on the force and frequency of contraction for muscle strips and for the effect on amplitude of the plateau and frequency of the action potential for single cells. CCK39 and CCK-OP had similar potencies and both forms were more potent than CCK33.

The inhibitory effects of vasoactive intestinal polypeptide on the mechanical and electrical activity of canine antral smooth muscle.

1. The inhibitory effects of vasoactive intestinal polypeptide (VIP) on the electrical and mechanical activity of canine antral smooth muscle were investigated. 2. In concentrations ranging from 5 X 10(-9) to 1 X 10(-7) M, VIP decreased the force of spontaneous contractions but had no measurable effect on spontaneous action potential complexes. 3. VIP had no effect on the increase in the amplitude and duration of the plateau potential and on the amplitude of contraction caused by a maximally effective concentration of acetylcholine. 4. VIP caused a significant decrease in the force of contraction caused by ED50 and threshold concentrations of acetylcholine. However, VIP had no measurable effect on the increase in the size of the action potential plateau caused by either concentration of acetylcholine. 5. VIP antagonized the increase in the amplitude of the plateau potential and the force of contraction induced by pentagastrin. It had no consistent effect on the pentagastrin-induced increase in frequency. 6. The data indicate that VIP acts as an inhibitor in this tissue in two distinct ways. It uncouples electromechanical coupling during spontaneous and acetylcholine-induced electrical and mechanical activity, and antagonizes pentagastrin-induced increases in electrical and mechanical activities.