CART Peptides Differently Regulate Firing Rates and GABAergic Synaptic Inputs of DMV Neurons Innervating the Stomach Antrum and Cecum of Adult Male Rats.

BACKGROUND/AIM: Central administration of cocaine- and amphetamine-regulated transcript peptides (CARTp) alters gastrointestinal motility and reduces food intake in rats. Since neurons in the dorsal motor nucleus of the vagus (DMV) receive GABAergic and glutamatergic inputs and innervate the smooth muscle of gastrointestinal organs, we hypothesized that CARTp acts on the DMV or presynaptic neurons. METHODS: We used 3,3'-dioctadecyloxa-carbocyanine perchlorate (DiO) retrograde tracing with electrophysiological methods to record DMV neurons innervating the stomach antrum or cecum in brainstem slices from adult rats. RESULTS: DiO application did not change the electrophysiological properties of DMV neurons. CART55-102 had no effect on the basal firing rates of neurons in either the stomach antrum-labeled group (SLG) or cecum-labeled group (CLG). When presynaptic inputs were blocked, CART55-102 further increased the firing rates of the SLG, suggesting a direct excitatory effect. Spontaneous inhibitory postsynaptic currents (sIPSCs) occurred at a higher frequency in SLG neurons than in CLG neurons. CART55-102 reduced the amplitude and the frequency of sIPSCs in SLG neurons dose-dependently, with higher doses also reducing spontaneous excitatory postsynaptic currents (sEPSCs). Higher doses of CART55-102 reduced sIPSC and sEPSC amplitudes in CLG neurons, suggesting a postsynaptic effect. In response to incremental current injections, the SLG neurons exhibited less increases in firing activity. Simultaneous applications of current injections and CART55-102 decreased the firing activity of the CLG. Therefore, stomach antrum-projecting DMV neurons possess a higher gating ability to stabilize firing activity. CONCLUSION: The mechanism by which CARTp mediates anorectic actions may be through a direct reduction in cecum-projecting DMV neuron excitability and, to a lesser extent, that of antrum-projecting DMV neurons, by acting on receptors of these neurons.

Abundance and Significance of Neuroligin-1 and Neurexin II in the Enteric Nervous System of Embryonic Rats.

Aim. To investigate the abundance of neuroligin-1 and neurexin II in the enteric nervous system (ENS) of rats on different embryonic days and to explore their potential significance. Methods. The full-thickness colon specimens proximal to the ileocecal junction of rats on embryonic days 16, 18, and 20 and of newborns within 24 hours (E16, E18, E20, and Ep0) were studied, respectively. qRT-PCR was applied for detecting the expressions of neuroligin-1 and neurexin II on mRNA, and western blotting was employed for detecting their further expressions on the whole tissue. Finally, the histological appearance of neuroligin-1 and neurexin IIα was elucidated using immunohistochemical staining. Results. qRT-PCR showed that the neuroligin-1 and neurexin II mRNA expressions of groups E16, E18, E20, and Ep0 increased gradually with the growth of embryonic rats (P < 0.05). Western blotting confirmed the increasing tendency. In immunohistochemical staining, proteins neuroligin-1 and neurexin IIα positive cells concentrated mostly in the myenteric nerve plexus of the colon and their expressions depend on the embryonic time. Conclusion. Neuroligin-1 and neurexin II were both expressed in the ENS and have temporal correlation with the development of ENS, during which neuronal intestinal malformations (NIM) may occur due to their disruptions and consequent abnormal ENS development.

The effect of gut microbiome on tolerance to morphine mediated antinociception in mice.

There is growing appreciation for the importance of gastrointestinal microbiota in many physiological and pathophysiological processes. While morphine and other narcotics are the most widely prescribed therapy for moderate to severe pain clinically, they have been noted to alter microbial composition and promote bacterial translocation to other tissues. Here we examined the pharmacodynamic properties of chronic morphine in mice following bacterial depletion with oral gavage of an antibiotic cocktail (ABX). ABX significantly reduced gut bacteria and prevented chronic morphine induced increases in gut permeability, colonic mucosal destruction, and colonic IL-1ß expression. In addition, ABX prevented the development of antinociceptive tolerance to chronic morphine in both the tail-immersion and acetic acid stretch assays. Morphine tolerance was also reduced by oral vancomycin that has 0% bioavailability. These findings were recapitulated in primary afferent neurons isolated from dorsal root ganglia (DRG) innervating the lower gastrointestinal tract, wherein in-vivo administration of ABX prevented tolerance to morphine-induced hypoexcitability. Finally, though ABX repeatedly demonstrated an ability to prevent tolerance, we show that it did not alter susceptibility to precipitation of withdrawal by naloxone. Collectively, these finding indicate that the gastrointestinal microbiome is an important modulator of physiological responses induced by chronic morphine administration.

Immunohistochemical distribution of cocaine and amphetamine regulatory peptide-like immunoreactive (CART-LI) nerve fibers in the circular muscle layer and their relationship to other peptides in the human caecum.

Motor activity of the gastrointestinal tract is extensively controlled by the enteric nervous system (ENS). Numerous neurotransmitters and neuromodulators are responsible for this regulation. One of them is cocaine- and amphetamine-regulated transcript peptide (CART). So far, there are few reports available concerning the distribution, functions, and co-localization of CART in the human gastrointestinal tract. The aim of the present investigation was to study the distribution and degree of co-localization of CART with substances taking part in conducting sensory stimuli, such as: substance P (SP), neurokinin A (NKA), calcitonin gene related peptide (CGRP) and Leu 5 enkephalin (L-ENK) in the circular muscle layer of the human caecum. CART-like immunoreactive (CART-LI) nerve fibers formed a very dense meshwork in the circular muscle layer of the caecum in all patients studied. Moreover, all neuronal substances tested during the present investigation were observed in CART-LI processes, but the degree of co-localization depended on the type of substance. The highest number of CART-positive nerves also contained L-ENK. A slightly lower level of co-localization was observed in the case of CART and SP or NKA, while only single nerve fibers were simultaneously CART- and CGRP-positive.

Luminal trypsin induces enteric nerve-mediated anion secretion in the mouse cecum.

Proteases play a diverse role in health and disease. An excessive concentration of proteases has been found in the feces of patients with inflammatory bowel disease or irritable bowel syndrome and been implicated in the pathogenesis of such disorders. This study examined the effect of the serine protease, trypsin, on intestinal epithelial anion secretion when added to the luminal side. A mucosal-submucosal sheet of the mouse cecum was mounted in Ussing chambers, and the short-circuit current (I sc) was measured. Trypsin added to the mucosal (luminal) side increased I sc with an ED50 value of approximately 10 µM. This I sc increase was suppressed by removing Cl(-) from the bathing solution. The I sc increase induced by 10-100 µM trypsin was substantially suppressed by tetrodotoxin, and partially inhibited by a neurokinin-1 receptor antagonist, but not by a muscarinic or nicotinic ACh-receptor antagonist. The trypsin-induced I sc increase was also significantly inhibited by a 5-hydroxytryptamine-3 receptor (5-HT3) antagonist and substantially suppressed by the simultaneous addition of both 5-HT3 and 5-HT4 receptor antagonists. We conclude that luminal trypsin activates the enteric reflex to induce anion secretion, 5-HT and substance P playing important mediating roles in this secreto-motor reflex. Luminal proteases may contribute to the cause of diarrhea occurring with some intestinal disorders.

Diabetic neuropathy: an evaluation of the use of quercetin in the cecum of rats.

AIM: To investigate the effect of quercetin supplementation on the myenteric neurons and glia in the cecum of diabetic rats. METHODS: Total preparations of the muscular tunic were prepared from the ceca of twenty-four rats divided into the following groups: control (C), control supplemented with quercetin (200 mg/kg quercetin body weight) (CQ), diabetic (D) and diabetic supplemented with quercetin (DQ). Immunohistochemical double staining technique was performed with HuC/D (general population)/nitric oxide synthase (nNOS), HuC-D/S-100 and VIP. Density analysis of the general neuronal population HuC/D-IR, the nNOS-IR (nitrergic subpopulation) and the enteric glial cells (S-100) was performed, and the morphometry and the reduction in varicosity population (VIP-IR) in these populations were analyzed. RESULTS: Diabetes promoted a significant reduction (25%) in the neuronal density of the HuC/D-IR (general population) and the nNOS-IR (nitrergic subpopulation) compared with the C group. Diabetes also significantly increased the areas of neurons, glial cells and VIP-IR varicosities. Supplementation with quercetin in the DQ group prevented neuronal loss in the general population and increased its area (P < 0.001) and the area of nitrergic subpopulation (P < 0.001), when compared to C group. Quercetin induced a VIP-IR and glial cells areas (P < 0.001) in DQ group when compared to C, CQ and D groups. CONCLUSION: In diabetes, quercetin exhibited a neuroprotective effect by maintaining the density of the general neuronal population but did not affect the density of the nNOS subpopulation.

Toxoplasma gondii infection causes morphological changes in caecal myenteric neurons.

The aim of this study was to evaluate the effects of chronic infection of Toxoplasma gondii (with genotype I and genotype III strains) on the population density and morphometry of caecal myenteric neurons in rats. Fifteen, 60-day-old, male Wistar rats (Rattus norvegicus) were used. The animals were assigned into three groups: Control Group (CG), Experimental Group 1 (EG1) and Experimental Group 2 (EG2). EG1 animals received 10(5) tachyzoites of the genotype I (BTU IV) T. gondii strain orally, and the EG2 animals received 10(5) tachyzoites of the genotype III (BTU II) strain orally. Thirty days after inoculation, caecal whole-mount preparations were stained by Giemsa technique. The caecal preparations were then analysed by assessing the population density and morphometry of myenteric neurons in specific regions of the caecum: mesenteric apical (MA), antimesenteric apical (AA), antimesenteric basal (AB) and next to caecal ampulla (NA). Myenteric neurons from the AA region were more clustered in EG1 animals (P<0.05). The EG1 animals presented a 16.8% reduction in the area of the nucleus, whereas the EG2 animals showed 18.4% increase (P<0.05). There was a more marked reduction in the cytoplasm of the animals in EG1 (↓23.2%) compared to EG2 (↓6.2%). There was 35.8% neuronal atrophy in the AB region and 16.8% in the region NA of the EG1 animals (P<0.05). In conclusion, different strains of T. gondii cause morphometric changes in caecal myenteric neurons of rats. Only the genotype I strain was able to cause neuronal density changes.

Effects of denervation at ileocecal junction and ileocecal resection in dogs.

BACKGROUND: To investigate neural regulation at the ileocecal junction (ICJ) and motility changes after ileocecal resection (ICR). Previous studies showed normal basal motility at the ICJ directly by force transducers in dogs, but these observations were limited to normal contractile activity. METHODS: Continuous strain gauge recordings of stomach, terminal ileum, ileocecal sphincter (ICS), and colon were performed in dogs. The dogs were divided into four groups, namely control (CONT), extrinsic denervation at ICJ (ED), intrinsic denervation at ICJ (ID), and ICR groups. Colonic activity was recorded 2 h before a meal, in the early postprandial period (first 2 h), and in the late postprandial period (4-6 h after a meal). The meal lasted 5 min. KEY RESULTS: Motility index was significantly increased at the ICS (P = 0.0056) and proximal colon (P = 0.0059) after feeding. However, such changes were not observed in the ED and ID groups. The amplitude of contractions at proximal colon in the interdigestive state was significantly decreased by ED. In the ID and ICR groups, the numbers of nonmigrating contractions were significantly decreased (P < 0.05), and colonic migrating motor complex (CMMC) ratio was significantly higher than that of the CONT group (P < 0.001). The dogs in these two groups had diarrhea. CONCLUSIONS & INFERENCES: Gastrocolonic response at the ICJ may require both intrinsic and extrinsic innervation. When ID was performed, CMMC ratio increased. As a result, intraluminal water absorption may have decreased. ID may be one of the causes of diarrhea after ICR.

Interstitial cells of Cajal in the gastrointestinal musculature of W(jic) c-kit mutant mice.

Interstitial cells of Cajal (ICC) generate electrical rhythmicity and transduce neural signals in the gastrointestinal musculature. ICC express the proto-oncogene c-kit, a receptor tyrosine kinase, and are identified morphologically by c-Kit immunoreactivity. The c-kit gene is allelic with the murine white-spotting locus W, and mutations of c-kit are known as W mutations. W mutations affect various developmental aspects of hematopoietic cells, germ cells, melanocytes, mast cells and ICC. We examined W(jic)/W(jic) mutant mice that have a mutation in the tyrosine kinase domain resulting in severe loss of protein function. W(jic)/W(jic) homozygotes exhibited white coats and black eyes. The gross morphology of the gastrointestinal tract showed no abnormality in mutant mice other than a forestomach papilloma. In the stomach, intramuscular ICC (ICC-IM) were missing, and myenteric ICC (ICC-MY) were reduced in number. In the small intestine, the number of ICC-MY was severely reduced; however there was a normal distribution of deep muscular plexus ICC (ICC-DMP). In the cecum, the numbers of ICC-IM and ICC-MY were severely depleted. ICC-IM were almost entirely absent in the colon, whereas ICC-MY loss was restricted to the distal colon. Patterns of ICC deficiency were generally similar between W(jic)/W(jic) mice and W/W(v) mutants, which lack a specific type of ICC. The enteric nervous system of the mutant mice appeared normal. From these findings, we conclude that W(jic)/W(jic) mice represent a distinct, novel genotype resulting in a lack of a specific type of ICC in the gastrointestinal musculature.

Colon electrical stimulation: potential use for treatment of obesity.

Obesity is one of the most prevalent health problems in the United States. Current therapeutic strategies for the treatment of obesity are unsatisfactory. We hypothesized the use of colon electrical stimulation (CES) to treat obesity by inhibiting upper gastrointestinal motility. In this preliminary study, we aimed at studying the effects of CES on gastric emptying of solid, intestinal motility, and food intake in dogs. Six dogs, equipped with serosal colon electrodes and a jejunal cannula, were randomly assigned to receive sham-CES or CES during the assessment of: (i) gastric emptying of solids, (ii) postprandial intestinal motility, (iii) autonomic functions, and (iv) food intake. We found that (i) CES delayed gastric emptying of solids by 77%. Guanethidine partially blocked the inhibitory effect of CES on solid gastric emptying; (ii) CES significantly reduced intestinal contractility and the effect lasted throughout the recovery period; (iii) CES decreased vagal activity in both fasting and fed states, increased the sympathovagal balance and marginally increased sympathetic activity in the fasting state; (iv) CES resulted in a reduction of 61% in food intake. CES reduces food intake in healthy dogs and the anorexigenic effect may be attributed to its inhibitory effects on gastric emptying and intestinal motility, mediated via the autonomic mechanisms. Further studies are warranted to investigate the therapeutic potential of CES for obesity.

Transient receptor potential ankyrin 1 is expressed by inhibitory motoneurons of the mouse intestine.

BACKGROUND & AIMS: Transient receptor potential ankyrin (TRPA) 1, an excitatory ion channel expressed by sensory neurons, mediates somatic and visceral pain in response to direct activation or noxious mechanical stimulation. Although the intestine is routinely exposed to irritant alimentary compounds and inflammatory mediators that activate TRPA1, there is no direct evidence for functional TRPA1 receptors on enteric neurons, and the effects of TRPA1 activation on intestinal function have not been determined. We characterized expression of TRPA1 by enteric neurons and determined its involvement in the control of intestinal contractility and transit. METHODS: TRPA1 expression was characterized by reverse-transcription polymerase chain reaction and immunofluorescence analyses. TRPA1 function was examined by Ca(2+) imaging and by assays of contractile activity and transit. RESULTS: We detected TRPA1 messenger RNA in the mouse intestine and TRPA1 immunoreactivity in enteric neurons. The cecum and colon had immunoreactivity for neuronal TRPA1, but the duodenum did not. TRPA1 immunoreactivity was also detected in inhibitory motoneurons and descending interneurons, cholinergic neurons, and intrinsic primary afferent neurons. TRPA1 activators, including cinnamaldehyde, allyl isothiocyanate (AITC), and 4-hydroxynonenal, increased [Ca(2+)](i) in myenteric neurons. These were reduced by a TRPA1 antagonist (HC-030031) or deletion of Trpa1. TRPA1 activation inhibited contractility of the segments of colon but not stomach or small intestine of Trpa1(+/+) but not Trpa1(-/-) mice; this effect was reduced by tetrodotoxin or N(G)-nitro-l-arginine methyl ester. Administration of AITC by gavage did not alter gastric emptying or small intestinal transit, but luminal AITC inhibited colonic transit via TRPA1. CONCLUSIONS: Functional TRPA1 is expressed by enteric neurons, and activation of neuronal TRPA1 inhibits spontaneous neurogenic contractions and transit of the colon.

Effects of L-glutamine supplementation on the myenteric neurons from the duodenum and cecum of diabetic rats.

CONTEXT: Peripheral neuropathy is one of the chronic complications of diabetes mellitus and is directly related to gastrointestinal consequences of the disease. Myenteric neurons are affected in some pathological conditions such as diabetic neuropathy. The imbalance between cellular antioxidants and free radicals, leading to an increase in oxidative stress, is considered one of the main factors responsible for neuronal damages in diabetes. Drugs that reduce the oxidative stress may play a significant role in the treatment of neurological complications of diabetes mellitus. OBJECTIVE: To evaluate the effect of L-glutamine supplementation on the myenteric neurons from the cecum and duodenum of Wistar rats with streptozotocin-induced diabetes mellitus. METHODS: The animals were divided in four groups (n = 5): non-treated normoglycemics, normoglycemics treated with L-glutamine, non-treated diabetics and diabetics treated with L-glutamine from the 4th day of diabetes induction on. The amino acid L-glutamine was added to their diet at 1%. Giemsa's technique was employed to stain the myenteric neurons. We determined the cell body area of 500 neurons in each group studied. The quantitative analysis was performed by sampling in an area of 16.6 mm² in the cecum and 3.6 mm² in the duodenum of each animal. RESULTS: After the supplementation with L-glutamine in the duodenum, we observed a preservation of neuronal density in groups normoglycemic and diabetic (P<0.05). We also observed a preservation of the cell bodies area in diabetic animals (group treated with L-glutamine) (P<0.05). In the cecum, that preservation was not evident. CONCLUSION: Supplementation with L-glutamine (1%) promoted a neuroprotective effect on the myenteric neurons from the duodenum of rats, both in terms of natural aging and of diabetes mellitus.

Effects of L-glutamine supplementation on the myenteric neurons from the duodenum and cecum of diabetic rats / Efeitos da suplementação com L-glutamina sobre os neurônios mioentéricos do duodeno e jejuno de ratos diabéticos

CONTEXT: Peripheral neuropathy is one of the chronic complications of diabetes mellitus and is directly related to gastrointestinal consequences of the disease. Myenteric neurons are affected in some pathological conditions such as diabetic neuropathy. The imbalance between cellular antioxidants and free radicals, leading to an increase in oxidative stress, is considered one of the main factors responsible for neuronal damages in diabetes. Drugs that reduce the oxidative stress may play a significant role in the treatment of neurological complications of diabetes mellitus. OBJECTIVE: To evaluate the effect of L-glutamine supplementation on the myenteric neurons from the cecum and duodenum of Wistar rats with streptozotocin-induced diabetes mellitus. METHODS: The animals were divided in four groups (n = 5): non-treated normoglycemics, normoglycemics treated with L-glutamine, non-treated diabetics and diabetics treated with L-glutamine from the 4th day of diabetes induction on. The amino acid L-glutamine was added to their diet at 1 percent. Giemsa's technique was employed to stain the myenteric neurons. We determined the cell body area of 500 neurons in each group studied. The quantitative analysis was performed by sampling in an area of 16.6 mm² in the cecum and 3.6 mm² in the duodenum of each animal. RESULTS: After the supplementation with L-glutamine in the duodenum, we observed a preservation of neuronal density in groups normoglycemic and diabetic (P<0.05). We also observed a preservation of the cell bodies area in diabetic animals (group treated with L-glutamine) (P<0.05). In the cecum, that preservation was not evident. CONCLUSION: Supplementation with L-glutamine (1 percent) promoted a neuroprotective effect on the myenteric neurons from the duodenum of rats, both in terms of natural aging and of diabetes mellitus.

CONTEXTO: Os neurônios entéricos são afetados em condições patológicas, como a neuropatia diabética. A neuropatia periférica é uma das complicações crônicas do diabetes mellitus e está diretamente relacionada com as manifestações gastrointestinais da doença. O desequilíbrio entre antioxidantes celulares e radicais livres, com o consequente aumento do estresse oxidativo, é considerado um dos principais responsáveis pelas alterações neuronais provocadas pelo diabetes. Drogas que reduzem o estresse oxidativo podem ter papel relevante no tratamento das complicações neurológicas do diabetes mellitus. OBJETIVO: Avaliar os efeitos da suplementação com L-glutamina sobre os neurônios mioentéricos do ceco e duodeno de ratos Wistar com diabetes mellitus induzido pela estreptozootocina. MÉTODOS: Os animais foram divididos em quatro grupos (n = 5): normoglicêmicos, normoglicêmicos suplementados com L-glutamina, diabéticos, diabéticos suplementados com L-glutamina a partir do 4º dia da indução do diabetes. O aminoácido L-glutamina foi adicionado à ração na quantidade de 1 por cento. A técnica de Giemsa foi utilizada para evidenciar os neurônios mioentéricos. Foram avaliadas as áreas de corpos celulares de 500 neurônios em cada grupo estudado. A análise quantitativa foi realizada em uma área de 16,6 mm² no ceco e 3,6 mm² no duodeno de cada animal. RESULTADOS: Após suplementação com L-glutamina verificou-se no duodeno a preservação da densidade neuronal tanto nos animais normoglicêmicos quanto nos animais diabéticos (P<0,05), e também o restabelecimento da área do corpo celular nos animais diabéticos (P<0,05). No ceco esta preservação e restabelecimento não foram evidenciados. CONCLUSÃO: A suplementação com L-glutamina (1 por cento) teve efeito neuroprotetor sobre os neurônios mioentéricos do duodeno tanto em condições de envelhecimento natural como no diabetes mellitus.

Pathological changes caused by Anoplocephala perfoliata in the mucosa/submucosa and in the enteric nervous system of equine ileocecal junction.

In this study, pathological changes caused by Anoplocephala perfoliata in the ileocecal junction were investigated in 31 regularly slaughtered mixed-breed horses of both sexes. Our results showed a significant relationship between parasite burden and grading of histopathological lesions in the mucosa and submucosa. Hypertrophy of the circular muscle layer was found in infected horses. Moreover, enteric nervous system evaluation showed a significant injury of intestinal nervous elements in the horses with moderate to high parasitism expressed as an increase of degenerative-regressive changes in neuronal cells and a decrease in the number of myenteric ganglia and neuronal cells. These findings can help to clarify the pathogenesis of intestinal motility disorders associated with A. perfoliata infection in horses.

Entamoeba histolytica infection and secreted proteins proteolytically damage enteric neurons.

The enteric protozoan parasite Entamoeba histolytica causes amebic colitis through disruption of the mucus layer, followed by binding to and destruction of epithelial cells. However, it is not known whether ameba infections or ameba components can directly affect the enteric nervous system. Analysis of mucosal innervations in the mouse model of cecal amebiasis showed that axon density was diminished to less than 25% of control. To determine whether amebas directly contributed to axon loss, we tested the effect of either E. histolytica secreted products (Eh-SEC) or soluble components (Eh-SOL) to an established coculture model of myenteric neurons, glia, and smooth muscle cells. Neuronal survival and axonal degeneration were measured after 48 h of exposure to graded doses of Eh-SEC or Eh-SOL (10 to 80 µg/ml). The addition of 80 µg of either component/ml decreased the neuron number by 30%, whereas the axon number was decreased by 50%. Cytotoxicity was specific to the neuronal population, since the glial and smooth muscle cell number remained similar to that of the control, and was completely abrogated by prior heat denaturation. Neuronal damage was partially prevented by the cysteine protease inhibitor E-64, showing that a heat-labile protease was involved. E. histolytica lysates derived from amebas deficient in the major secreted protease EhCP5 caused a neurotoxicity similar to that of wild-type amebas. We conclude that E. histolytica infection and ameba protease activity can cause selective damage to enteric neurons.

Sympathetic innervation of the ileocecal junction in horses.

The distribution and chemical phenotypes of sympathetic and dorsal root ganglion (DRG) neurons innervating the equine ileocecal junction (ICJ) were studied by combining retrograde tracing and immunohistochemistry. Immunoreactivity (IR) for tyrosine hydroxylase (TH), dopamine beta-hydroxylase (DBH), neuronal nitric oxide synthase (nNOS), calcitonin gene-related peptide (CGRP), substance P (SP), and neuropeptide Y (NPY) was investigated. Sympathetic neurons projecting to the ICJ were distributed within the celiac (CG), cranial mesenteric (CranMG), and caudal mesenteric (CaudMG) ganglia, as well as in the last ganglia of the thoracic sympathetic chain and in the splanchnic ganglia. In the CG and CranMG 91 +/- 8% and 93 +/- 12% of the neurons innervating the ICJ expressed TH- and DBH-IR, respectively. In the CaudMG 90 +/- 15% and 94 +/- 5% of ICJ innervating neurons were TH- and DBH-IR, respectively. Sympathetic (TH-IR) fibers innervated the myenteric and submucosal ganglia, ileal blood vessels, and the muscle layers. They were more concentrated at the ICJ level and were also seen encircling myenteric plexus (MP) and submucosal plexus (SMP) descending neurons that were retrogradely labeled from the ICJ. Among the few retrogradely labeled DRG neurons, nNOS-, CGRP-, and SP-IR nerve cells were observed. Dense networks of CGRP-, nNOS-, and SP-IR varicosities were seen around retrogradely labeled prevertebral ganglia neurons. The CGRP-IR fibers are probably the endings of neurons projecting from the intestine to the prevertebral ganglia. These findings indicate that this crucial region of the intestinal tract is strongly influenced by the sympathetic system and that sensory information of visceral origin influences the sympathetic control of the ICJ.

Analysis of myenteric neurons of the cecum of diabetic rats after supplementation with ascorbic acid / Análisis de neuronas mientéricas del ciego de ratas diabéticas después de suplementación con ácido ascórbico

The objective of this work was to investigate the neuroprotective action of the ascorbic acid over the myenteric neurons in the cecum of Wistar rats, four months after induction of the diabetes mellitus experimental with streptozotocin. Three groups with five rats each were used: C- controls, D- diabetic, DA- diabetic treated with ascorbic acid. For evidentiation of the myenteric neurons was carried out to Giemsa's technique. Were evaluated the areas of cell bodies of 500 neurons in each group studied. The quantitative analysis was carried out in an area of 16.6 mm2 in each cecum studied. In the animals diabetic observed elevation of the glycemia and glycated hemoglobin. The supplementation with ascorbic acid was effective under the myenteric neurons of the cecum of diabetics rays, since was presented the effect neuroprotective and neurotrofic.

El objetivo de este trabajo fue verificar el efecto neuroprotector del ácido ascórbico sobre las neuronas mientéricas en el ciego de Rattus Wistar, cuatro meses después de la inducción de diabetes mellitus experimental con estreptozotocina. Utilizamos tres grupos de animales: C- control, D- diabético, DA- diabético tratado con ácido ascórbico. Para la observación de las neuronas mientéricas fue llevado a cabo la técnica de Giemsa. Fueron evaluadas las áreas del soma de 500 neuronas, en cada grupo estudiado. El análisis cuantitativo fue llevado a cabo, en cada ciego, en un área de 16,6 mm². En los animales diabéticos, se observó la elevación de la glicemia y de la hemoglobina glicosilada. La suplementación con ácido ascórbico fue efectiva en las neuronas mientéricas del ciego de animales diabéticos, ya que se produjeron los efectos neuroprotetor y neurotrófico.

Intrinsic innervation of the ileocaecal junction in the horse: preliminary study.

REASON FOR PERFORMING STUDY: In horses, morpho-functional studies related to the enteric nervous system (ENS) controlling the sphincters are lacking. OBJECTIVES: To investigate immunohistochemically the morphology, distribution, density, phenotypes and projections of neurons controlling the ileocaecal junction (ICJ). METHODS: Two young horses were anaesthetised and underwent midline laparotomy. The neuronal retrograde fluorescent tracer Fast Blue (FB) was injected into the wall of the ICJ. A post surgical survival time of 30 days was used. Following euthanasia, the ileum and a small portion of caecum were removed. Cryosections were used to investigate the immunoreactivity (IR) of the neurons innervating the ICJ for choline acetyltransferase (ChAT), neuronal nitric oxide synthase (nNOS), substance P (SP), calcitonin gene-related peptide (CGRP) and neurofilament NF200kDa (NF). RESULTS: Ileal FB-labelled neurons innervating the ICJ were located in the myenteric plexus (MP) and submucosal plexus (SMP) up to 48 cm and 28 cm, respectively, from the point of the FB injections. Descending MP and SMP neurons were nitrergic (54 +/- 11% and 68 +/- 4%, respectively), cholinergic (60 +/- 19% and 82 +/- 11%, respectively), NF-IR (54 +/- 9% and 78 +/- 21%, respectively), and SP-IR (about 20% in both the plexuses). CGRP-IR was expressed only by SMP descending neurons (45 +/- 21%). In both the plexuses descending neurons coexpressing nNOS- and ChAT-IR were also observed (25 +/- 11% and 61 +/- 27%, respectively). CONCLUSIONS: The presence of ileal long projecting neurons innervating the ICJ suggests that they are critical for its modulation. Consequently, in bowel diseases in which the resection of the terminal jejunum and proximal ileum are required, it is preferable, whenever possible, to conserve the major portion of the ileum. POTENTIAL RELEVANCE: The knowledge of the phenotype of ENS neurons of the ileum might be helpful for developing pharmaceutical treatment of the ICJ motility disorders.

Inhibitory purinergic transmission in mouse caecum: role for P2Y1 receptors as prejunctional modulators of ATP release.

Using conventional microelectrode recording techniques, we investigated, in the circular muscle of the mouse caecum, the neurotransmitter(s) involved in the neurally-evoked inhibitory junction potentials (IJPs) and the existence of possible prejunctional mechanisms controlling neurotransmitter release. Electrical field stimulation with single pulses elicited IJPs, consisting only of a "fast" hyperpolarization, while using train stimuli (30-50 Hz) the initial fast hyperpolarization was followed by a slower hyperpolarization. The fast and the slow component were selectively antagonized by apamin, a blocker of calcium-activated potassium channels, and N(omega)-nitro-l-arginine methyl ester (l-NAME), a nitric oxide synthase inhibitor, respectively. Fast IJPs were antagonized also by P2 purinoceptor antagonists, suramin or 4-[[4-formyl-5-hydroxy-6-methyl-3-[(phosphonooxy)methyl]-2-pyridinyl]azo]-1,3-benzenedisulfonic acid tetrasodium salt (PPADS), P2Y purinoceptor desensitization by adenosine 5'-O-2-thiodiphosphate (ADPbetaS). 2'-Deoxy-N(6)-methyl ADP diammonium salt (MRS 2179), P2Y1 purinoceptor antagonist, at the concentration of 1 microM increased the amplitude of the fast IJP, while at the concentration of 10 microM induced a reduction. 8,8'-[Carbonylbis[imino-3,1-phenylenecarbonylimino (4-fluoro-3,1-phenylene) carbonylimino]] bis-1,3,5-naphthalenetrisulfonic acid hexasodium salt (NF 157) and 2,2-dimethyl-propionic acid 3-(2-chloro-6-methylaminopurin-9-yl)-2-(2,2-dimethyl-propionyl-oxymethyl)-propyl ester (MRS 2395), P2Y11 and P2Y12 purinoceptor antagonist, were without any effect. ATP-induced hyperpolarization was affected by apamin and by P2Y purinoceptor desensitization, but not by MRS 2179. 2-(Methylthio)ATP tetrasodium salt hydrate (2-MeSATP), P2Y1 purinoceptor agonist, at a concentration which did not cause changes in the membrane potential, reduced the amplitude of the fast IJPs. This effect was prevented by MRS 2179. Paired nerve stimulation, either using single pulses or train stimuli, did not cause any alteration of the second-evoked IJP. In conclusion, in the circular muscle of the mouse caecum, ATP is responsible for the fast IJP while nitric oxide is responsible for the slow IJP. ATP-mediated response is dependent on ADPbetaS-sensitive P2Y receptors, which are in part P2Y1, but not P2Y11 or P2Y12 receptor subtypes. In addition, the most substantial finding of this study is the functional demonstration that ATP released by nerve stimulation activates P2Y1 receptors, located prejunctionally, limiting its release by motoneurons.

Functional effects and characteristics of cecum-projecting neurons in the dorsal motor nucleus of the vagus of rats.

Preganglionic neurons in the dorsal motor nucleus of the vagus (DMV) innervate most of the gastrointestinal tract; with the stomach and the cecum/proximal colon having a greater proportion of vagal input. Cecum-projecting neurons have been thought to be distinct from other preganglionic neurons due to their location within the DMV, but it is unknown whether these neurons innervate the cecum exclusively or what effect their activation has on cecal motor activity. Therefore, we investigated the extent of coinnervation of cecum and stomach by vagal neurons, their neurochemistry, and the effect of DMV stimulation on intracecal and intragastric volumes. Fluorescent retrograde tracers injected into the serosa of the cecum and stomach revealed that in the DMV 49+/-5% CTB-labeled cecum-projecting neurons also innervated the stomach. Immunocytochemical staining for nitric oxide (NO) synthase and tyrosine hydroxylase indicated that only 3+/-1% and 4+/-1% of cecum-projecting neurons contained these markers, respectively. In anesthetized rats gastric and cecal volumes were measured by prototypic miniaturized dual barostats that were developed for use in rodents. Microinjection of l-glutamate into the DMV increased gastric contractile activity and tone, and reduced on-going cecum contractile activity (2.6+/-0.7 contractions/2 min after injection versus 8.2+/-0.4 contractions/2 min before injection, N = 5). The barostat was able to detect decreases (-0.88+/-0.13 ml) and increases (0.25+/-0.05 ml) in cecum volume in response to carbachol and sodium nitroprusside, respectively. In summary, cecum-projecting neurons are not an entirely exclusive population within the DMV because a percentage of these also innervate the stomach. Central vagal stimulation can modulate both gastric and cecum contractile activity. Together, these data support a role of the vagus in neural reflexes involving gastric and large bowel motor function, such as the immediate phase of the gastrocolonic reflex.