Changes of excitatory and inhibitory neurotransmitters in the colon of rats underwent to the wrap partial restraint stress.

BACKGROUND: Animal models proposed to reproduce some of the human irritable bowel syndrome (IBS) symptoms are based on the hypothesis that psychosocial stressors play a pivotal role in the IBS etio-pathology. We investigated the wrap restraint stress (WRS) model with the aim to analyze the morphological changes of the entire colonic wall of these animals that showed some of the human IBS symptoms such as visceral hypersensitivity. METHODS: Male Wistar rats were used and WRS was maintained for 2 h. Abdominal contractions (AC) were recorded in the colon-rectum by balloon distension. Fecal pellets were quantitated. Colonic specimens were examined by routine histology, immunohistochemistry and western blot. KEY RESULTS: WRS animals were characterized by: (i) increase in AC number and fecal pellets mean weight; (ii) clusters of mononucleated cells, increase in eosinophilic granulocytes and mast cells in the mucosa; (iii) increase in CGRP-immunoreactive (IR) nerve fibers in the lamina propria; (iv) decrease in myenteric NK1r-IR and nNOS-IR neurons and in submucous nNOS-IR neurons; (v) decrease in SP-IR nerve fibers in the muscle wall; (vi) reduction in S100ß-IR glia in the entire colonic wall; (vii) increase in CRF1r-IR myenteric neurons; (viii) no change in ChAT-IR neurons, smooth muscle cells and interstitial cells of Cajal. CONCLUSIONS AND INFERENCES: The present results support the consistency of the WRS as a potential model where part of the human IBS signs and symptoms are reproduced. The changes in glial cells and in excitatory and inhibitory neurotransmitters might represent the substrate for the dysmotility and hypersensitivity.

Changes in nitrergic and tachykininergic pathways in rat proximal colon in response to chronic treatment with otilonium bromide.

BACKGROUND: Otilonium bromide (OB) is used as a spasmolytic drug in the treatment of the functional bowel disorder irritable bowel syndrome. Although its acute effects on colonic relaxation are well-characterized, little is known about the effects of chronic administration of OB on enteric neurons, neuromuscular transmission, and interstitial cells of Cajal (ICC), key regulators of the gut function. METHODS: Adult Sprague-Dawley rats were treated with OB in drinking water at a dose of 2 mg/kg for 30 days. The colons of OB-treated and age-matched control rats were studied by confocal immunohistochemistry to detect immunoreactivity (IR) in myenteric plexus neurons for nitrergic and tachykininergic markers, and also by microelectrode electrophysiology. KEY RESULTS: Using immunohistochemistry, chronic OB administration did not change total neuron number, assessed by anti-Hu IR, but resulted in a significant increase in NK1 receptor positive neurons, a decrease in neuronal nitric oxide synthase expressing neurons, and a reduction in volume of substance P in nerve fibers in the myenteric plexus. Chronic OB administration potentiated inhibitory and excitatory junction potentials evoked by repetitive electrical field stimulation. The various types of colonic ICC, detected by Kit IR, were not altered nor were slow waves or smooth muscle membrane potential. CONCLUSIONS & INFERENCES: Chronic treatment with OB caused significant changes in the nitrergic and tachykinergic components of the myenteric plexus and in both inhibitory and excitatory neurotransmission in the rat colon.

Chronic treatment with otilonium bromide induces changes in L-type Ca²âº channel, tachykinins, and nitric oxide synthase expression in rat colon muscle coat.

BACKGROUND: Otilonium bromide (OB) is a quaternary ammonium derivative used for the treatment of intestinal hypermotility and is endowed with neurokinin2 receptor (NK2r) antagonist and Ca²âº channel blocker properties. Therefore, the possibility that OB might play a role in the neurokinin receptor/Substance-P/nitric oxide (NKr/SP/NO) circuit was investigated after chronic exposition to the drug. METHODS: Rats were treated with OB 2-20 mg kg⁻¹ for 10 and 30 days. In the proximal colon, the expression and distribution of muscle NOsynthase 1 (NOS1), NK1r, NK2r, SP and Cav 1.2 subunit (for L-type Ca²âº channel) and the spontaneous activity and stimulated responses to NK1r and NK2r agonists were investigated. KEY RESULTS: Immunohistochemistry showed a redistribution of NK1r and L-type Ca²âº channel in muscle cells with no change of NK2r at 30 days, a significant increase in muscle NOS1 expression at 10 days and a significant decrease in the SP content early in the ganglia and later in the intramuscular nerve fibers. Functional studies showed no change in spontaneous activity but a significant increase in maximal contraction induced by NK1r agonist. CONCLUSIONS & INFERENCES: Chronic exposition to OB significantly affects the NKr/SP/NO circuit. The progressive decrease in SP-expression might be the consequence of the persistent presence of OB, the increase of NOS1 expression in muscle cells at 10 days in an attempt to guarantee an adequate NO production, and, at 30 days, the redistribution of the L-type Ca²âº channel and NK1r as a sign to compensate the drug channel block by re-cycling both of them. The physiological data suggest NK1r hypersensitivity.

Clinical-histological associations in gastroparesis: results from the Gastroparesis Clinical Research Consortium.

BACKGROUND: Cellular changes associated with diabetic (DG) and idiopathic gastroparesis (IG) have recently been described from patients enrolled in the Gastroparesis Clinical Research Consortium. The association of these cellular changes with gastroparesis symptoms and gastric emptying is unknown. The aim of this study was to relate cellular changes to symptoms and gastric emptying in patients with gastroparesis. METHODS: Earlier, using full thickness gastric body biopsies from 20 DG, 20 IG, and 20 matched controls, we found decreased interstitial cells of Cajal (ICC) and enteric nerves and an increase in immune cells in both DG and IG. Here, demographic, symptoms [gastroparesis cardinal symptom index score (GCSI)], and gastric emptying were related to cellular alterations using Pearson's correlation coefficients. KEY RESULTS: Interstitial cells of Cajal counts inversely correlated with 4 h gastric retention in DG but not in IG (r = -0.6, P = 0.008, DG, r = 0.2, P = 0.4, IG). There was also a significant correlation between loss of ICC and enteric nerves in DG but not in IG (r = 0.5, P = 0.03 for DG, r = 0.3, P = 0.16, IG). Idiopathic gastroparesis with a myenteric immune infiltrate scored higher on the average GCSI (3.6 ± 0.7 vs 2.7 ± 0.9, P = 0.05) and nausea score (3.8 ± 0.9 vs 2.6 ± 1.0, P = 0.02) as compared to those without an infiltrate. CONCLUSIONS & INFERENCES: In DG, loss of ICC is associated with delayed gastric emptying. Interstitial cells of Cajal or enteric nerve loss did not correlate with symptom severity. Overall clinical severity and nausea in IG is associated with a myenteric immune infiltrate. Thus, full thickness gastric biopsies can help define specific cellular abnormalities in gastroparesis, some of which are associated with physiological and clinical characteristics of gastroparesis.

GLP-2 receptor expression in excitatory and inhibitory enteric neurons and its role in mouse duodenum contractility.

BACKGROUND: Glucagon-like peptide 2 (GLP-2), a nutrient-responsive hormone, exerts various actions in the gastrointestinal tract that are mediated by a G-protein coupled receptor called GLP-2R. A little information is available on GLP-2R expression in enteric neurons and nothing on the interstitial cells of Cajal (ICC). METHODS: We investigated presence and distribution of the GLP-2R in the mouse duodenum by immunohistochemistry and the potential motor effects of GLP-2 on the spontaneous and neurally evoked mechanical activity. KEY RESULTS: The GLP-2R was expressed by the myenteric and submucosal neurons. Labelling was also present in nerve varicosities within the circular muscular layer and at the deep muscular plexus (DMP). No immunoreactive nerve fiber was seen within the longitudinal muscle layer. The GLP-2R-positive neurons were either excitatory (SP- and choline-acetyltransferase-positive) or inhibitory (vasoactive intestinal polypeptide and nNOS-positive). The ICC, both at the myenteric plexus and at the DMP, never expressed GLP-2R but, especially those at the DMP, were surrounded by GLP-2R-positive nerve varicosities co-expressing either excitatory or inhibitory neurotransmitters. Quantitative analysis demonstrated a consistent prevalence of GLP-2R on the excitatory pathways. In agreement, the functional results showed that the administration of GLP-2 in vitro caused decrease of the spontaneous contractions mediated by nitric oxide release and reduction of the evoked cholinergic contractions. CONCLUSIONS & INFERENCES: The present findings indicate that the GLP-2R is expressed by inhibitory and excitatory neurons, the GLP-2 inhibits the muscle contractility likely decreasing cholinergic neurotransmission and increasing nitric oxide production, and this effect is possibly mediated by the ICC-DMP recruitment.

Effect of otilonium bromide and ibodutant on the internalization of the NK2 receptor in human colon.

BACKGROUND: The present aim was to study the modulation of NK2 receptor internalization by two compounds, the spasmolytic otilonium bromide (OB) endowed with NK2 receptor antagonistic properties and the selective NK2 receptor antagonist ibodutant. METHODS: Full-thickness human colonic segments were incubated in the presence of OB (0.1-10 µmol L(-1)) or ibodutant (0.001-0.1 µmol L(-1)), with or without the NK2 receptor selective agonist [ßAla8]NKA(4-10) and then fixed in 4% paraformaldehyde. Cryosections were processed for NK2 receptor immunohistochemical revelation. Quantitative analysis evaluated the number of the smooth muscle cells that had internalized the NK2 receptor. KEY RESULTS: Immunohistochemistry revealed that in basal condition, the NK2 receptor was internalized in about 23% of total smooth muscle cells. The exposure to the selective NK2 receptor agonist induced internalization of the receptor in more than 77% of the cells. Previous exposure to both OB or ibodutant, either alone or in the presence of the agonist, concentration-dependently reduced the number of the cells with the internalized receptor. CONCLUSIONS & INFERENCES: Both OB and ibodutant antagonize the internalization of the NK2 receptor in the human colon. As NK2 receptors are the predominant receptor mediating spasmogenic activity of tachykinins on enteric smooth muscle, we hypothesize that the antagonistic activity found for both OB and ibodutant should play a specific therapeutic role in gut diseases characterized by hypermotility.

Peripheral motor action of glucagon-like peptide-1 through enteric neuronal receptors.

BACKGROUND: Glucagon-like peptide-1 (GLP-1) is a proglucagon-derived peptide expressed in the enteroendocrine-L cells of small and large intestine and released in response to meal ingestion. Glucagon-like peptide-1 exerts inhibitory effects on gastrointestinal motility through vagal afferents and central nervous mechanisms; however, no data is available about a direct influence on the gastrointestinal wall. Our aim was to investigate the effects of GLP-1 on the spontaneous and evoked mechanical activity of mouse duodenum and colon and to identify the presence and distribution of GLP-1 receptors (GLP-1R) in the muscle coat. METHODS: Organ bath recording technique and immunohistochemistry were used. KEY RESULTS: Glucagon-like peptide-1 (up to the concentration of 1 mumol L(-1)) failed to affect spontaneous mechanical activity. It caused concentration-dependent reduction of the electrically evoked cholinergic contractions in circular smooth muscle of both intestinal segments, without affecting the longitudinal muscle responses. Glucagon-like peptide-1 inhibitory effect was significantly antagonized by exendin (9-39), an antagonist of GLP-1R. In both intestinal preparations, GLP-1 effect was not affected by guanethidine, a blocker of adrenergic neurotransmission, but it was significantly reduced by N(omega)-nitro-l-arginine methyl ester, inhibitor of nitric oxide (NO) synthase. Glucagon-like peptide-1 failed to affect the contractions evoked by exogenous carbachol. Immunohistochemistry demonstrated GLP-1R expression in the enteric neurons. Furthermore, 27% of GLP-1R immunoreactive (IR) neurons in the duodenum and 79% of GLP-1R-IR neurons in the colon, co-expressed nNOS. CONCLUSIONS & INFERENCES: The present results suggest that GLP-1 is able to act in the enteric nervous system by decreasing the excitatory cholinergic neurotransmission through presynaptic GLP-1Rs, which modulate NO release.

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.

Interstitial Cajal-like cells in rat mesentery: an ultrastructural and immunohistochemical approach.

Interstitial Cajal-like Cells (ICLC) were recently recognized in a plethora of non-digestive organs. Here, we describe a cell type of rat mesentery sharing ultrastructural and immunohistochemical features with ICLC. Mesenteric ICLC were demonstrated by transmission electron microscopy (TEM) and further tested by light microscope immunohistochemistry. The cell described here fulfils the TEM diagnostic criteria accepted for ICLC: location in the connective interstitium; close vicinity to nerves, capillaries and other interstitial cells; characteristic long, moniliform cell processes; specialized cell-to-cell junctions; caveolae; mitochondria at 5-10% of cytoplasmic volume; rough endoplasmic reticulum at about 1-2%; intermediate and thin filaments, microtubules; undetectable thick filaments. The processes of this mesenteric ICLC were particularly long, with a mean length of 24.91 microm (10.27-50.83 micorm), and a convolution index of 2.32 (1.37-3.63) was calculated in order to measure their potential length. Mean distances versus main target cells of ICLC-nerve bundles, vessels, adipocytes and macrophages-were 110.69, 115.80, 205.07 and 34.65 nm, respectively. We also tested the expression of CD117/c-kit, CD34, vimentin, alpha-smooth muscle actin, nestin, NK-1, tryptase and chymase and the antigenic profile of the mesenteric ICLC was comparable if not identical with that recently observed in ICLC from other extra-digestive tissues. Due to the peculiar aspect of the mesenteric ICLC processes it can be hypothesized that these cells form a three-dimensional network within the mesentery that is at the same time resistant and deformable following stretches consequent to intestine movements, mainly avoiding blood vessels closure or controlling blood vessels rheology. It remains, however, to be established if and how such cells are connected with the archetypal enteric ICC.

Altered tachykinergic influence on gastric mechanical activity in mdx mice.

This study investigated whether alterations in gastric activity in dystrophic mdx mouse can be attributed to dysfunctions of tachykinins. Endoluminal pressure was recorded and the expression of neuronal nitric oxide synthase (nNOS), NK1 and NK2 neurokinin receptors was investigated by immunohistochemistry. SR48968, NK2 receptor antagonist, but not SR140333, NK1 receptor antagonist, decreased the tone only in mdx gastric preparations. In the presence of N(omega)-nitro-l-arginine methyl ester (l-NAME), inhibitor of NOS, SR48968 reduced the tone also in normal stomach. [Sar(9), Met(O(2))(11)]-SP, agonist of NK1 receptors, caused tetrodotoxin-sensitive relaxations, antagonized by SR140333 or l-NAME, with no difference in the potency or efficacy between normal and mdx preparations. [beta-Ala(8)]-NKA(4-10), an NK2 receptor agonist, induced SR48968-sensitive contractions in both types of preparations, although the maximal response of mdx tissues was significantly lower than normal preparations. Immunohistochemistry demonstrated a consistent reduction of nNOS and NK2 receptor expression in mdx stomach smooth muscle cells and no change in nNOS and NK1 receptor expression in neurones. In conclusion, in mdx stomach the activation of NK2 receptors plays a role in the development of the tone, associated with a reduced NO production by muscular nNOS. The hypo-responsiveness to NK2 receptors could depend on the reduced expression of these receptors.

Expression of neuronal and inducible nitric oxide synthase in neuronal and glial cells after transient occlusion of the middle cerebral artery.

We presently investigated the time-course of neuronal nitric oxide synthase and inducible nitric oxide synthase expression and content in the rat striatum up to 6 days after ischemia induced by transient middle cerebral artery occlusion, a condition that potentially allows functional recovery, with the aim to identify the cell types expressing these two enzymes and to correlate neuronal nitric oxide synthase and inducible nitric oxide synthase changes in order to verify whether and how these changes are related to tissue damage, motor-sensory performances and survival. Before and after surgery, the animals underwent neurological evaluation. The results demonstrated that the rats with a score > or = 12 at the neurological evaluation 24 h after ischemia showed a significant increase in neuronal nitric oxide synthase-immunoreactive neurones and absence of inducible nitric oxide synthase-immunoreactive cells and survived up to the sixth day; conversely, the rats with a score < 12 at the neurological evaluation 24 h after ischemia showed a progressive significant decrease in neuronal nitric oxide synthase-immunoreactive neurones and appearance of inducible nitric oxide synthase-immunoreactive cells and none of the rats survived up to the sixth day. Microglia cells were activated in both groups but only in the latter did these cells express inducible nitric oxide synthase. Measurement of the infarct area demonstrated that it occupied a similar territory in both groups of rats but in those with a score < 12 the edema was more extended. In conclusion, we demonstrated that a neurotoxic insult such as ischemia can induce neuronal nitric oxide synthase expression in the neurones and that when neuronal nitric oxide synthase-immunoreactive neurones increase in number, microglia activation is less extended, inducible nitric oxide synthase-immunoreactive cells are absent, tissue damage reduced and the rats survive longer. Conversely, when there is a significant decrease of neuronal nitric oxide synthase-immunoreactive neurones, microglia cells are intensely activated, inducible nitric oxide synthase-immunoreactive cells appear and the animal survival is shortened.

Gastroschisis in the rat model is associated with a delayed maturation of intestinal pacemaker cells and smooth muscle cells.

BACKGROUND: A pacemaker system is required for peristalsis generation. The interstitial cells of Cajal (ICC) are considered the intestinal pacemaker, and are identified by expression of the c-kit gene--encoded protein. Gastroschisis is characterized by a severe gastrointestinal dysmotility in newborns. In spite of this clinical picture, few studies have focused on smooth muscle cells (SMC) morphology and none on ICC. Therefore, their morphology has been studied in fetuses at term in the rat model of gastroschisis. METHODS: At 18.5 day's gestation (E18.5), 10 rat fetuses were killed, 10 underwent surgical creation of gastroschisis, and 10 underwent manipulation only. The small intestine of the latter 2 groups was harvested at E21.5. Specimens were processed for H&E, c-kit and actin (alpha smooth muscle antibody [alpha-SMA]) immunohistochemistry, and transmission electron microscopy (TEM). RESULTS: In the controls, SMC were c-kit+ and alpha-SMA+, with labeling intensity increasing by age. At E21.5, some cells around the Auerbach's plexus were more intensely c-kit+, and differentiating ICC were seen under TEM at this level. Gastroschisis fetuses had no c-kit+ cells referable to ICC. In the more damaged loops, SMC were very faintly c-kit+ and alpha-SMA+. Under TEM, there were few differentiated SMC and no presumptive ICC. In the less-damaged loops, SMC were faintly c-kit+ and alpha-SMA+ and had ultrastructural features intermediate between those of E18.5 and E21.5 controls; ICC were very immature. CONCLUSIONS: ICC and SMC differentiation is delayed in gastroschisis with the most damaged loops showing the most incomplete picture. These findings might help in understanding the delayed onset of peristalsis and the variable time-course of the recover seen in babies affected by gastroschisis.

Interstitital cells of Cajal in the human stomach: distribution and relationship with enteric innervation.

Interstitial cells of Cajal (ICC) are distributed throughout the gastrointestinal muscle coat with a region-specific location, and are considered to be pace-maker and/or mediators of neurotransmission. Little is known about their shape, size, distribution and relationships with excitatory and inhibitory nerves in human stomach. With this aim, we labeled the ICC, using c-Kit immunohistochemistry, followed by a quantitative analysis to evaluate the distribution and area occupied by these cells in the circular and longitudinal muscle layers and at the myenteric plexus level in the human fundus, corpus and antrum. Furthermore, by NADPH-d histochemistry and substance P (SP) immunohistochemistry, we labeled and quantified nitric oxide (NO)-producing and SP-containing nerves and evidenced their relationships with the ICC in these three gastric regions. In the fundus, the ICC appeared as bipolar cells and in the corpus and antrum they mainly appeared as multipolar cells, with highly ramified processes. The networks formed by ICC differed in the three gastric regions. The ICC number was significantly higher and cell area smaller in the fundus compared to the corpus and antrum. The area occupied by the ICC was significantly higher at the myenteric plexus level compared with circular and longitudinal muscle layers. Everywhere, NADPH-d-positive nerves were more numerous than SP-positive ones. Both kinds of fibers were closely apposed to the ICC in the corpus and antrum. In conclusion, in the human stomach, the ICC have region-specific shape, size and distribution and in the corpus and antrum have close contact with both inhibitory and excitatory nerves. Presumably, as suggested for laboratory mammals, these differences are in relationship with the motor activities peculiar to each gastric area.

Neuronal differentiation and myenteric plexus organization are delayed in gastroschisis: an immunohistochemical study in a rat model.

Gastroschisis is a malformation due to prenatal rupture of the abdominal wall and evisceration of the midgut. Intestinal loops are shortened, matted, and covered by a peel caused by the harmful effect of the amniotic fluid. Babies born with gastroschisis suffer from gastrointestinal dysmotility. The present aim was to verify whether the myenteric plexus is damaged in a rat model of gastroschisis. In the gastroschisis rat model fetus, the myenteric plexus was not yet organized in the well-defined ganglia and, in the most damaged loops, the neuronal cells were scattered or absent. Immunohistochemistry for alpha-internexin and peripherin (markers of neuronal maturity) gave results similar to those of earlier embryonic ages. These findings indicate a delay in neuronal differentiation and myenteric plexus organization that might play a role in the postnatal dysmotility observed in gastroschisis.

Myenteric neurons and interstitial cells of Cajal of mouse colon express several nitric oxide synthase isoforms.

Information on equipment and subcellular distribution of nitric oxide synthase (NOS) isoforms in myenteric neurons and pacemaker cells (ICC) might help to identify nitric oxide (NO) pathway(s) acting on gastrointestinal motility. In sections of mouse colon labelled with neuronal (n)NOS, endothelial (e)NOS and inducible (i)NOS antibodies, all myenteric neurons co-expressed eNOS and iNOS and a subpopulation of them co-expressed nNOS. ICC co-expressed nNOS and eNOS. In the neurons, nNOS-labeling was intracytoplasmatic, in the ICC at cell periphery. In both cell types, eNOS-labeling was on intracytoplasmatic granules, likely mitochondria. In conclusion, myenteric neurons and ICC co-express several NOS isoforms with specific subcellular distribution. Different nNOS splice variants are presumably present: intracytoplasmatic nNOSbeta and nNOSalpha producing neurogenic NO, plasma membrane-bound nNOSalpha producing ICCgenic NO. eNOS might be implicated in mitochondrial respiration and, in ICC, also in pacemaker activity. Neurons express iNOS also in basal condition.

Alterations of neurokinin receptors and interstitial cells of Cajal during and after jejunal inflammation induced by Nippostrongylus brasiliensis in the rat.

Substance P (SP) and its receptors NK1 and NK2 are widely expressed in the intestinal wall by neurones, interstitial cells of Cajal (ICC) and smooth muscle cells. Changes in SP and/or its NK receptors have been documented during experimental inflammation in animals or inflammatory bowel diseases in humans, but the data concern the acute phase of the inflammatory process. We determined immunohistochemically whether NK receptors and SP were altered in the muscle coat during jejunal inflammation induced by the nematode Nippostrongylus brasiliensis and whether these alterations persisted when inflammation had spontaneously resolved 30 days postinfection. An ultrastructural analysis was also conducted on ICC, nerves and muscle. At day 14, when inflammation peaked, there was a reduction in NK1 receptors in myenteric neurones and in SP-immunoreactive nerve endings. There were also ultrastructural anomalies in synaptic vesicles and NK2 receptor loss in the circular muscle layer. The SP decrease persisted at day 30, whereas neurones and circular muscle cells re-expressed NK1 and NK2 receptors, respectively. The ICC at the deep muscular plexus, located near to the inflammatory site, underwent alterations leading to their complete loss at day 30. These morphological changes are probably associated with impairment in tachykinergic control of jejunal functions leading to the alterations of motility and sensitivity to distension already described in these animals.

Myogenic NOS and endogenous NO production are defective in colon from dystrophic (mdx) mice.

The aim of the present study was to evaluate whether alterations in the distribution and/or function of nitric oxide synthase (NOS) could be involved in the development of the spontaneous mechanical tone observed in colon from dystrophic (mdx) mice. By recording the intraluminal pressure of isolated colon from normal mice, we showed that N(omega)-nitro- L-arginine methyl ester (L-NAME) increased the tone, even in the presence of tetrodotoxin. The effect was prevented by L-arginine, nifedipine, or Ca(2+)-free solution. In colon from mdx mice, L-NAME was ineffective. Immunohistochemistry revealed that the presence and distribution of neuronal (nNOS), endothelial, and inducible NOS isoforms in smooth muscle cells and neurons of colon from mdx mice were the same as in controls. However, the expression of myogenic nNOS was markedly reduced in mdx mice. We conclude that there is a myogenic NOS in mouse colon that can tonically produce nitric oxide to limit influx of Ca(2+) through L-type voltage-dependent channels and modulate the mechanical tone. This mechanism appears to be defective in mdx mice.

Expression of dystrophin in the mouse myenteric neurones.

Dystrophin, a membrane-associated protein, plays relevant roles in cell functions. Its lack or trunkated expression results in Duchenne muscular dystrophy (DMD), a pathology associated with alterations in gastrointestinal motility considered to be neural in origin. No data are available on the presence of dystrophin in myenteric neurones. We labelled mouse myenteric neurones with DYS1-, DYS2-, DYS3-antibodies; staining was located on the perikarya and processes, with no differences in distribution or intensity among the antibodies; the western immunoblot analysis indicated that myenteric neurones express several dystrophin isoforms; anti-dystrophins/anti-neuronal specific enolase double-labeling confirmed that all neurones express dystrophin. Dystrophin in myenteric neurones might play a role in cytoskeletal organization, axonal transport and signal pathways; its lack might cause the intestinal motor abnormalities reported in DMD patients.

Synapse formation during neuron differentiation: an in situ study of the myenteric plexus during murine embryonic life.

Ultrastructural steps characterizing synapse formation in vivo and appearance in neuroblasts of properties suggestive of synaptic function acquisition have scarcely been studied. Synapse formation and proteosynthetic apparatus organization were thus studied under transmission electron microscope in mouse myenteric neurons from embryonic day 12.5 (E12.5) until birth. Expression of Ret and p75(NTR), markers of neural crest cells, as well as that of neuron-specific enolase (NSE), synaptophysin (SY), and synaptosomal-associated protein (SNAP), markers of synaptic function acquisition, were immunohistochemically evaluated. At E12.5 many cells were Ret- and p75(NTR)-immunoreactive (IR), whereas a few were NSE-IR and had neuronal ultrastructural characteristics. Two types of contacts between poorly or nondifferentiated cells and axons of presumed extrinsic (synapse-like contacts) or local (immature synapses) origin were identified, along with SY-IR elements. By E16. 5, many cells had developed a proteosynthetic apparatus, synapse-like contacts were no longer present, and immature synapses were gradually differentiating. Concurrently, there was an increase in NSE-IR cells, some of which were also SNAP-IR, and in SY-IR varicosities. At E18.5, ultrastructurally mature neurons and synapses had increased in number as had NSE-IR and SNAP-IR cells and SY-IR varicosities. These data indicate that 1) one type of contact (synapse-like) is present at E12.5 between very immature cells and presumed vagal fibers, with a possible transient role for the onset of the differentiative process of these cells; and 2) another type of contact (typical synapses) lasts until E18.5, with a similar but long-lasting role that progressively shifts to the classical function (neurotransmission) as the synapse matures and the embryo reaches the day of birth.

NK1, NK2 and NK3 tachykinin receptor localization and tachykinin distribution in the ileum of the mouse.

Tachykinin receptors NK1r, NK2r and NK3r bind tachykinins with different affinities and share pharmacological and molecular differences among animal species. NK1r, NK2r, NK3r and tachykinin (SP/NKA) distribution was studied by immunohistochemistry in the ileum of mouse since no data are available for this species. The results were then compared to those obtained in the rat and guinea pig either by us or by others to ascertain interspecies similarities and/or differences. NK1r- and NK3r-immunoreactivity (IR) were detected in neurons and NK1r-IR in the interstitial cells of Cajal at the deep muscular plexus. At variance with rat and guinea pig, NK1r-IR was also found in the myoid cells of the villi, while NK2r-IR was never detected in nerve varicosities. This latter datum suggests that the NK2r does not play a presynaptic role in the mouse. Unexpectedly, a high NK2r-IR and the presence of NK3r-IR were observed at the inner portion of the circular muscle layer in the mouse as well as in the rat and guinea pig, demonstrating a subregional distribution of these receptors. Tachykinin distribution did not show noticeable species-related differences. The present findings show species-related differences in the tachykinin receptor distribution that might be related to a different tachykinin control of intestinal motility.